Schmidt M

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Full name : Schmidt Monika

First name : Monika

Mail : Biomedical Research Centre , University Hospital Hradec Kralove , Sokolska 581 , 500 05 Hradec Kralove

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Country : Czech Republic

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References (62)

Title : A-series agent A-234: initial in vitro and in vivo characterization - Hrabinova_2024_Arch.Toxicol__
Author(s) : Hrabinova M , Pejchal J , Hepnarova V , Muckova L , Junova L , Opravil J , Zdarova Karasova J , Rozsypal T , Dlabkova A , Rehulkova H , Kucera T , Vecera Z , Caisberger F , Schmidt M , Soukup O , Jun D
Ref : Archives of Toxicology , : , 2024
Abstract : A-series agent A-234 belongs to a new generation of nerve agents. The poisoning of a former Russian spy Sergei Skripal and his daughter in Salisbury, England, in March 2018 led to the inclusion of A-234 and other A-series agents into the Chemical Weapons Convention. Even though five years have already passed, there is still very little information on its chemical properties, biological activities, and treatment options with established antidotes. In this article, we first assessed A-234 stability in neutral pH for subsequent experiments. Then, we determined its inhibitory potential towards human recombinant acetylcholinesterase (HssAChE; EC 3.1.1.7) and butyrylcholinesterase (HssBChE; EC 3.1.1.8), the ability of HI-6, obidoxime, pralidoxime, methoxime, and trimedoxime to reactivate inhibited cholinesterases (ChEs), its toxicity in rats and therapeutic effects of different antidotal approaches. Finally, we utilized molecular dynamics to explain our findings. The results of spontaneous A-234 hydrolysis showed a slow process with a reaction rate displaying a triphasic course during the first 72 h (the residual concentration 86.2%). A-234 was found to be a potent inhibitor of both human ChEs (HssAChE IC(50) = 0.101 +/- 0.003 microM and HssBChE IC(50) = 0.036 +/- 0.002 microM), whereas the five marketed oximes have negligible reactivation ability toward A-234-inhibited HssAChE and HssBChE. The acute toxicity of A-234 is comparable to that of VX and in the context of therapy, atropine and diazepam effectively mitigate A-234 lethality. Even though oxime administration may induce minor improvements, selected oximes (HI-6 and methoxime) do not reactivate ChEs in vivo. Molecular dynamics implies that all marketed oximes are weak nucleophiles, which may explain the failure to reactivate the A-234 phosphorus-serine oxygen bond characterized by low partial charge, in particular, HI-6 and trimedoxime oxime oxygen may not be able to effectively approach the A-234 phosphorus, while pralidoxime displayed low interaction energy. This study is the first to provide essential experimental preclinical data on the A-234 compound.
ESTHER : Hrabinova_2024_Arch.Toxicol__
PubMedSearch : Hrabinova_2024_Arch.Toxicol__
PubMedID: 38446233

Title : Non-covalent acetylcholinesterase inhibitors: In vitro screening and molecular modeling for novel selective insecticides - Hepnarova_2022_Toxicol.In.Vitro__105463
Author(s) : Hepnarova V , Hrabinova M , Muckova L , Kucera T , Schmidt M , Dolezal R , Gorecki L , Hrabcova V , Korabecny J , Mezeiova E , Jun D , Pejchal J
Ref : Toxicol In Vitro , :105463 , 2022
Abstract : Insecticides represent the most crucial element in the integrated management approach to malaria and other vector-borne diseases. The evolution of insect resistance to long-used substances and the toxicity of organophosphates (OPs) and carbamates are the main factors contributing to the development of new, environmentally safe pesticides. In our work, fourteen compounds of 7-methoxytacrine-tacrine heterodimers were tested for their insecticidal effect. Compounds were evaluated in vitro on insect acetylcholinesterase from Anopheles gambiae (AgAChE) and Musca domestica (MdAChE). The evaluation was executed in parallel with testing on human erythrocyte acetylcholinesterase (HssAChE) and human butyrylcholinesterase (HssBChE) using a modified Ellman's method. Compound efficacy was determined as IC(50) values for the respective enzymes and selectivity indexes were expressed to compare the interspecies selectivity. Docking studies were performed to predict the binding modes of selected compounds. K1328 and K1329 provided high HssAChE/AgAChE selectivity outperforming standard pesticides (carbofuran and bendiocarb), and thus can be considered as suitable lead structure for novel anticholinesterase insecticides.
ESTHER : Hepnarova_2022_Toxicol.In.Vitro__105463
PubMedSearch : Hepnarova_2022_Toxicol.In.Vitro__105463
PubMedID: 36041654

Title : Charged pyridinium oximes with thiocarboxamide moiety are equally or less effective reactivators of organophosphate-inhibited cholinesterases compared to analogous carboxamides - Kohoutova_2022_J.Enzyme.Inhib.Med.Chem_37_760
Author(s) : Kohoutova Z , Malinak D , Andrys R , Svobodova J , Psotka M , Schmidt M , Prchal L , Musilek K
Ref : J Enzyme Inhib Med Chem , 37 :760 , 2022
Abstract : The organophosphorus antidotes, so-called oximes, are able to restore the enzymatic function of acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) via cleavage of organophosphate from the active site of the phosphylated enzyme. In this work, the charged pyridinium oximes containing thiocarboxamide moiety were designed, prepared and tested. Their stability and pK(a) properties were found to be analogous to parent carboxamides (K027, K048 and K203). The inhibitory ability of thiocarboxamides was found in low microM levels for AChE and high microM levels for BChE. Their reactivation properties were screened on human recombinant AChE and BChE inhibited by nerve agent surrogates and paraoxon. One thiocarboxamide was able to effectively restore function of NEMP- and NEDPA-AChE, whereas two thiocarboxamides were able to reactivate BChE inhibited by all tested organophosphates. These results were confirmed by reactivation kinetics, where thiocarboxamides were proved to be effective, but less potent reactivators if compared to carboxamides.
ESTHER : Kohoutova_2022_J.Enzyme.Inhib.Med.Chem_37_760
PubMedSearch : Kohoutova_2022_J.Enzyme.Inhib.Med.Chem_37_760
PubMedID: 35193448

Title : Pyridinium-2-carbaldoximes with quinolinium carboxamide moiety are simultaneous reactivators of acetylcholinesterase and butyrylcholinesterase inhibited by nerve agent surrogates - Lee_2021_J.Enzyme.Inhib.Med.Chem_36_437
Author(s) : Lee HM , Andrys R , Jonczyk J , Kim K , Vishakantegowda AG , Malinak D , Skarka A , Schmidt M , Vaskova M , Latka K , Bajda M , Jung YS , Malawska B , Musilek K
Ref : J Enzyme Inhib Med Chem , 36 :437 , 2021
Abstract : The pyridinium-2-carbaldoximes with quinolinium carboxamide moiety were designed and synthesised as cholinesterase reactivators. The prepared compounds showed intermediate-to-high inhibition of both cholinesterases when compared to standard oximes. Their reactivation ability was evaluated in vitro on human recombinant acetylcholinesterase (hrAChE) and human recombinant butyrylcholinesterase (hrBChE) inhibited by nerve agent surrogates (NIMP, NEMP, and NEDPA) or paraoxon. In the reactivation screening, one compound was able to reactivate hrAChE inhibited by all used organophosphates and two novel compounds were able to reactivate NIMP/NEMP-hrBChE. The reactivation kinetics revealed compound 11 that proved to be excellent reactivator of paraoxon-hrAChE better to obidoxime and showed increased reactivation of NIMP/NEMP-hrBChE, although worse to obidoxime. The molecular interactions of studied reactivators were further identified by in silico calculations. Molecular modelling results revealed the importance of creation of the pre-reactivation complex that could lead to better reactivation of both cholinesterases together with reducing particular interactions for lower intrinsic inhibition by the oxime.
ESTHER : Lee_2021_J.Enzyme.Inhib.Med.Chem_36_437
PubMedSearch : Lee_2021_J.Enzyme.Inhib.Med.Chem_36_437
PubMedID: 33467931

Title : Amaryllidaceae Alkaloids of Norbelladine-Type as Inspiration for Development of Highly Selective Butyrylcholinesterase Inhibitors: Synthesis, Biological Activity Evaluation, and Docking Studies - Al Mamun_2021_Int.J.Mol.Sci_22_
Author(s) : Al Mamun A , Pidany F , Hulcova D , Marikova J , Kucera T , Schmidt M , Catapano MC , Hrabinova M , Jun D , Muckova L , Kunes J , Janousek J , Andrys R , Novakova L , Perinova R , Maafi N , Soukup O , Korabecny J , Cahlikova L
Ref : Int J Mol Sci , 22 : , 2021
Abstract : Alzheimer's disease (AD) is a multifactorial neurodegenerative condition of the central nervous system (CNS) that is currently treated by cholinesterase inhibitors and the N-methyl-d-aspartate receptor antagonist, memantine. Emerging evidence strongly supports the relevance of targeting butyrylcholinesterase (BuChE) in the more advanced stages of AD. Within this study, we have generated a pilot series of compounds (1-20) structurally inspired from belladine-type Amaryllidaceae alkaloids, namely carltonine A and B, and evaluated their acetylcholinesterase (AChE) and BuChE inhibition properties. Some of the compounds exhibited intriguing inhibition activity for human BuChE (hBuChE), with a preference for BuChE over AChE. Seven compounds were found to possess a hBuChE inhibition profile, with IC(50) values below 1 microM. The most potent one, compound 6, showed nanomolar range activity with an IC(50) value of 72 nM and an excellent selectivity pattern over AChE, reaching a selectivity index of almost 1400. Compound 6 was further studied by enzyme kinetics, along with in-silico techniques, to reveal the mode of inhibition. The prediction of CNS availability estimates that all the compounds in this survey can pass through the blood-brain barrier (BBB), as disclosed by the BBB score.
ESTHER : Al Mamun_2021_Int.J.Mol.Sci_22_
PubMedSearch : Al Mamun_2021_Int.J.Mol.Sci_22_
PubMedID: 34361074

Title : Derivatives of montanine-type alkaloids and their implication for the treatment of Alzheimer's disease: Synthesis, biological activity and in silico study - Maafi_2021_Bioorg.Med.Chem.Lett_51_128374
Author(s) : Maafi N , Pidany F , Marikova J , Korabecny J , Hulcova D , Kucera T , Schmidt M , Shammari LA , Spulak M , Carmen Catapano M , Mecava M , Prchal L , Kunes J , Janousek J , Kohelova E , Jenco J , Novakova L , Cahlikova L
Ref : Bioorganic & Medicinal Chemistry Lett , 51 :128374 , 2021
Abstract : Alzheimes disease (AD) is the most common neurodegenerative disorder, characterized by neuronal loss and cognitive impairment. Currently, very few drugs are available for AD treatment, and a search for new therapeutics is urgently needed. Thus, in the current study, twenty-eight new derivatives of montanine-type Amaryllidaceae alkaloids were synthesized and evaluated for their ability to inhibit human recombinant acetylcholinesterase (hAChE) and butyrylcholinesterase (hBuChE). Three derivatives (1n, 1o, and 1p) with different substitution patterns demonstrated significant selective inhibitory potency for hAChE (IC(50) < 5 microM), and one analog, 1v, showed selective hBuChE inhibition activity (IC(50) = 1.73 +/- 0.05 microM). The prediction of CNS availability, as disclosed by the BBB score, suggests that the active compounds in this survey should be able pass through the blood-brain barrier (BBB). Cytotoxicity screening and docking studies were carried out for the two most pronounced cholinesterase inhibitors, 1n and 1v.
ESTHER : Maafi_2021_Bioorg.Med.Chem.Lett_51_128374
PubMedSearch : Maafi_2021_Bioorg.Med.Chem.Lett_51_128374
PubMedID: 34555506

Title : Acetylcholinesterase Inhibition of Diversely Functionalized Quinolinones for Alzheimer's Disease Therapy - Bautista-Aguilera_2020_Int.J.Mol.Sci_21_
Author(s) : Bautista-Aguilera OM , Ismaili L , Chioua M , Andrys R , Schmidt M , Bzonek P , Martinez-Grau MA , Beadle CD , Vetman T , Lopez-Munoz F , Iriepa I , Refouvelet B , Musilek K , Marco-Contelles J
Ref : Int J Mol Sci , 21 : , 2020
Abstract : In this communication, we report the synthesis and cholinesterase (ChE)/monoamine oxidase (MAO) inhibition of 19 quinolinones (QN1-19) and 13 dihydroquinolinones (DQN1-13) designed as potential multitarget small molecules (MSM) for Alzheimer's disease therapy. Contrary to our expectations, none of them showed significant human recombinant MAO inhibition, but compounds QN8, QN9, and DQN7 displayed promising human recombinant acetylcholinesterase (hrAChE) and butyrylcholinesterase (hrBuChE) inhibition. In particular, molecule QN8 was found to be a potent and quite selective non-competitive inhibitor of hrAChE (IC50 = 0.29 microM), with Ki value in nanomolar range (79 nM). Pertinent docking analysis confirmed this result, suggesting that this ligand is an interesting hit for further investigation.
ESTHER : Bautista-Aguilera_2020_Int.J.Mol.Sci_21_
PubMedSearch : Bautista-Aguilera_2020_Int.J.Mol.Sci_21_
PubMedID: 32486316

Title : Cysteine-Targeted Insecticides against A. gambiae Acetylcholinesterase Are Neither Selective nor Reversible Inhibitors - Gorecki_2020_ACS.Med.Chem.Lett_11_65
Author(s) : Gorecki L , Andrys R , Schmidt M , Kucera T , Psotka M , Svobodova B , Hrabcova V , Hepnarova V , Bzonek P , Jun D , Kuca K , Korabecny J , Musilek K
Ref : ACS Med Chem Lett , 11 :65 , 2020
Abstract : Acetylcholinesterase cysteine-targeted insecticides against malaria vector Anopheles gambia and other mosquitos have already been introduced. We have applied the olefin metathesis for the preparation of cysteine-targeted insecticides in high yields. The prepared compounds with either a succinimide or maleimide moiety were evaluated on Anopheles gambiae and human acetylcholinesterase with relatively high irreversible inhibition of both enzymes but poor selectivity. The concept of cysteine binding was not proved by several methods, and poor stability was observed of the chosen most potent/selective compounds in a water/buffer environment. Thus, our findings do not support the proposed concept of cysteine-targeted selective insecticides for the prepared series of succinimide or maleimide compounds.
ESTHER : Gorecki_2020_ACS.Med.Chem.Lett_11_65
PubMedSearch : Gorecki_2020_ACS.Med.Chem.Lett_11_65
PubMedID: 31938465

Title : LIPG-promoted lipid storage mediates adaptation to oxidative stress in breast cancer - Cadenas_2019_Int.J.Cancer_145_901
Author(s) : Cadenas C , Vosbeck S , Edlund K , Grgas K , Madjar K , Hellwig B , Adawy A , Glotzbach A , Stewart JD , Lesjak MS , Franckenstein D , Claus M , Hayen H , Schriewer A , Gianmoena K , Thaler S , Schmidt M , Micke P , Ponten F , Mardinoglu A , Zhang C , Kafferlein HU , Watzl C , Frank S , Rahnenfuhrer J , Marchan R , Hengstler JG
Ref : International Journal of Cancer , 145 :901 , 2019
Abstract : Endothelial lipase (LIPG) is a cell surface associated lipase that displays phospholipase A1 activity towards phosphatidylcholine present in high-density lipoproteins (HDL). LIPG was recently reported to be expressed in breast cancer and to support proliferation, tumourigenicity and metastasis. Here we show that severe oxidative stress leading to AMPK activation triggers LIPG upregulation, resulting in intracellular lipid droplet accumulation in breast cancer cells, which supports survival. Neutralizing oxidative stress abrogated LIPG upregulation and the concomitant lipid storage. In human breast cancer, high LIPG expression was observed in a limited subset of tumours and was significantly associated with shorter metastasis-free survival in node-negative, untreated patients. Moreover, expression of PLIN2 and TXNRD1 in these tumours indicated a link to lipid storage and oxidative stress. Altogether, our findings reveal a previously unrecognized role for LIPG in enabling oxidative stress-induced lipid droplet accumulation in tumour cells that protects against oxidative stress, and thus supports tumour progression.
ESTHER : Cadenas_2019_Int.J.Cancer_145_901
PubMedSearch : Cadenas_2019_Int.J.Cancer_145_901
PubMedID: 30653260

Title : Cognitive, emotional and social phenotyping of mice in an observer-independent setting - Dere_2018_Neurobiol.Learn.Mem_150_136
Author(s) : Dere E , Ronnenberg A , Tampe B , Arinrad S , Schmidt M , Zeisberg E , Ehrenreich H
Ref : Neurobiol Learn Mem , 150 :136 , 2018
Abstract : Based on the intellicage paradigm, we have developed a novel cognitive, emotional and social phenotyping battery that permits comprehensive standardized behavioral characterization of mice in an experimenter-independent social setting. Evaluation of this battery in a large number of male and female C57BL/6 wildtype mice, tested in >20 independent cohorts, revealed high reproducibility of the behavioral readouts and may serve as future reference tool. We noticed robust sex-specific differences in general activity, cognitive and emotional behavior, but not regarding preference for social pheromones. Specifically, female mice revealed higher activity, decreased sucrose preference, impaired reversal and place-time-reward learning. Furthermore, female mice reacted more sensitively than males to reward-withdrawal showing a negative emotional contrast/Crespi-effect. In a series of validation experiments, we tested mice with different pathologies, including neuroligin-3 deficient mice (male Nlgn3(y/-) and female Nlgn3(+/-)) for autistic behavior, oligodendrocyte-specific erythropoietin receptor knockout (oEpoR(-/-)) mice for cognitive impairment, as well as mouse models of renal failure (unilateral ureteral obstruction and 5/6 nephrectomy) and of type 2 diabetes (ApoE(-/-)) - for delineating potentially confounding effects of motivational factors (thirst, glucose-craving) on learning and memory assessments. As prominent features, we saw in Nlgn3 mutants reduced preference for social pheromones, whereas oEpoR(-/-) mice showed learning deficits in place or reversal learning tasks. Renal failure led to increased water intake, and diabetic metabolism to enhanced glucose preference, limiting interpretation of hereon based learning and memory performance in these mice. The phenotyping battery presented here may be well-suited as high-throughput multifaceted diagnostic instrument for translational neuropsychiatry and behavioral genetics.
ESTHER : Dere_2018_Neurobiol.Learn.Mem_150_136
PubMedSearch : Dere_2018_Neurobiol.Learn.Mem_150_136
PubMedID: 29474958

Title : A green tea polyphenol epigallocatechin-3-gallate enhances neuroregeneration after spinal cord injury by altering levels of inflammatory cytokines - Machova_2017_Neuropharmacol_126_213
Author(s) : Machova Urdzikova L , Ruzicka J , Karova K , Kloudova A , Svobodova B , Amin A , Dubisova J , Schmidt M , Kubinova S , Jhanwar-Uniyal M , Jendelova P
Ref : Neuropharmacology , 126 :213 , 2017
Abstract : Spinal cord injury (SCI) is a debilitating condition which is characterized by an extended secondary injury due to the presence of inflammatory local milieu. Epigallocatechin gallate (EGCG) appears to possess strong neuroprotective properties. Here, we evaluated the beneficial effect of EGCG on recovery from SCI. Male Wistar rats were given either EGCG or saline directly to the injured spinal cord and thereafter a daily IP injection. Behavior recovery was monitored by BBB, plantar, rotarod and flat-beam tests. The levels of inflammatory cytokines were determined on days 1, 3, 7, 10 and 14 after SCI. Additionally, NF-kappaB pathway activity was evaluated. The results demonstrated that EGCG-treated rats displayed a superior behavioral performance in a flat beam test, higher axonal sprouting and positive remodelation of glial scar. Cytokine analysis revealed a reduction in IL-6, IL2, MIP1alpha and RANTES levels on days 1 and 3, and an upregulation of IL-4, IL-12p70 and TNFalpha 1 day following SCI in EGCG-treated rats. Treatment with EGCG was effective in decreasing the nuclear translocation of subunit p65 (RelA) of the NF-kappaB dimer, and therefore canonical NF-kappaB pathway attenuation. A significant increase in the gene expression of growth factors (FGF2 and VEGF), was noted in the spinal cord of EGCG-treated rats. Further, EGCG influenced expression of M1 and M2 macrophage markers. Our results have demonstrated a therapeutic value of EGCG in SCI, as observed by better behavioral performance measured by flat beam test, modulation of inflammatory cytokines and induction of higher axonal sprouting.
ESTHER : Machova_2017_Neuropharmacol_126_213
PubMedSearch : Machova_2017_Neuropharmacol_126_213
PubMedID: 28899730

Title : The Janthinobacterium sp. HH01 genome encodes a homologue of the V. cholerae CqsA and L. pneumophila LqsA autoinducer synthases - Hornung_2013_PLoS.One_8_e55045
Author(s) : Hornung C , Poehlein A , Haack FS , Schmidt M , Dierking K , Pohlen A , Schulenburg H , Blokesch M , Plener L , Jung K , Bonge A , Krohn-Molt I , Utpatel C , Timmermann G , Spieck E , Pommerening-Roser A , Bode E , Bode HB , Daniel R , Schmeisser C , Streit WR
Ref : PLoS ONE , 8 :e55045 , 2013
Abstract : Janthinobacteria commonly form biofilms on eukaryotic hosts and are known to synthesize antibacterial and antifungal compounds. Janthinobacterium sp. HH01 was recently isolated from an aquatic environment and its genome sequence was established. The genome consists of a single chromosome and reveals a size of 7.10 Mb, being the largest janthinobacterial genome so far known. Approximately 80% of the 5,980 coding sequences (CDSs) present in the HH01 genome could be assigned putative functions. The genome encodes a wealth of secretory functions and several large clusters for polyketide biosynthesis. HH01 also encodes a remarkable number of proteins involved in resistance to drugs or heavy metals. Interestingly, the genome of HH01 apparently lacks the N-acylhomoserine lactone (AHL)-dependent signaling system and the AI-2-dependent quorum sensing regulatory circuit. Instead it encodes a homologue of the Legionella- and Vibrio-like autoinducer (lqsA/cqsA) synthase gene which we designated jqsA. The jqsA gene is linked to a cognate sensor kinase (jqsS) which is flanked by the response regulator jqsR. Here we show that a jqsA deletion has strong impact on the violacein biosynthesis in Janthinobacterium sp. HH01 and that a jqsA deletion mutant can be functionally complemented with the V. cholerae cqsA and the L. pneumophila lqsA genes.
ESTHER : Hornung_2013_PLoS.One_8_e55045
PubMedSearch : Hornung_2013_PLoS.One_8_e55045
PubMedID: 23405110
Gene_locus related to this paper: 9burk-l9pl81 , 9burk-l9pql3 , 9burk-l9pc92 , 9burk-l9pf07 , 9burk-l9pgi7 , 9burk-l9p8t0 , 9burk-l9pl28

Title : Use of 'small but smart' libraries to enhance the enantioselectivity of an esterase from Bacillus stearothermophilus towards tetrahydrofuran-3-yl acetate - Nobili_2013_FEBS.J_280_3084
Author(s) : Nobili A , Gall MG , Pavlidis IV , Thompson ML , Schmidt M , Bornscheuer UT
Ref : Febs J , 280 :3084 , 2013
Abstract : Two libraries of simultaneous double mutations in the active site region of an esterase from Bacillus stearothermophilus were constructed to improve the enantioselectivity in the hydrolysis of tetrahydrofuran-3-yl acetate. As screening of large mutant libraries is hampered by the necessity for GC/MS analysis, mutant libraries were designed according to a 'small but smart' concept. The design of focused libraries was based on data derived from a structural alignment of 3317 amino acid sequences of alpha/beta-hydrolase fold enzymes with the bioinformatic tool 3DM. In this way, the number of mutants to be screened was substantially reduced as compared with a standard site-saturation mutagenesis approach. Whereas the wild-type esterase showed only poor enantioselectivity (E = 4.3) in the hydrolysis of (S)-tetrahydrofuran-3-yl acetate, the best variants obtained with this approach showed increased E-values of up to 10.4. Furthermore, some variants with inverted enantiopreference were found.
ESTHER : Nobili_2013_FEBS.J_280_3084
PubMedSearch : Nobili_2013_FEBS.J_280_3084
PubMedID: 23331978

Title : Promiscuous enantioselective (-)-gamma-lactamase activity in the Pseudomonas fluorescens esterase I - Torres_2012_Org.Biomol.Chem_10_3388
Author(s) : Torres LL , Schliessmann A , Schmidt M , Silva-Martin N , Hermoso JA , Berenguer J , Bornscheuer UT , Hidalgo A
Ref : Org Biomol Chem , 10 :3388 , 2012
Abstract : A promiscuous but very enantioselective (-)-gamma-lactamase activity in the kinetic resolution of the Vince lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) was detected in the Pseudomonas fluorescens esterase I (PFEI). The lactamase activity was increased 200-fold by the introduction of a point mutation and resulted as enantioselective as the Microbacterium sp. enzyme used industrially in this resolution. The structural and mechanistic determinants for the catalytic promiscuity and enantioselectivity were identified by molecular modeling, setting a ground stone to engineer further amidase-related activities from this esterase.
ESTHER : Torres_2012_Org.Biomol.Chem_10_3388
PubMedSearch : Torres_2012_Org.Biomol.Chem_10_3388
PubMedID: 22359066
Gene_locus related to this paper: psefl-este

Title : The metagenome-derived enzymes LipS and LipT increase the diversity of known lipases - Chow_2012_PLoS.One_7_e47665
Author(s) : Chow J , Kovacic F , Dall Antonia Y , Krauss U , Fersini F , Schmeisser C , Lauinger B , Bongen P , Pietruszka J , Schmidt M , Menyes I , Bornscheuer UT , Eckstein M , Thum O , Liese A , Mueller-Dieckmann J , Jaeger KE , Streit WR
Ref : PLoS ONE , 7 :e47665 , 2012
Abstract : Triacylglycerol lipases (EC 3.1.1.3) catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75 degrees C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70 degrees C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70 degrees C. LipS had an optimum temperature at 70 degrees C and LipT at 75 degrees C. Both enzymes catalyzed hydrolysis of long-chain (C(12) and C(14)) fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R)-ibuprofen-phenyl ester with an enantiomeric excess (ee) of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70 degrees C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 A revealing an unusually compact lid structure.
ESTHER : Chow_2012_PLoS.One_7_e47665
PubMedSearch : Chow_2012_PLoS.One_7_e47665
PubMedID: 23112831
Gene_locus related to this paper: symth-q67mr3 , 9bact-k7qe48

Title : PMCA2 via PSD-95 controls calcium signaling by alpha7-containing nicotinic acetylcholine receptors on aspiny interneurons - Gomez-Varela_2012_J.Neurosci_32_6894
Author(s) : Gomez-Varela D , Schmidt M , Schoellerman J , Peters EC , Berg DK
Ref : Journal of Neuroscience , 32 :6894 , 2012
Abstract : Local control of calcium concentration within neurons is critical for signaling and regulation of synaptic communication in neural circuits. How local control can be achieved in the absence of physical compartmentalization is poorly understood. Challenging examples are provided by nicotinic acetylcholine receptors that contain alpha7 nicotinic receptor subunits (alpha7-nAChRs). These receptors are highly permeable to calcium and are concentrated on aspiny dendrites of interneurons, which lack obvious physical compartments for constraining calcium diffusion. Using functional proteomics on rat brain, we show that alpha7-nAChRs are associated with plasma membrane calcium-ATPase pump isoform 2 (PMCA2). Analysis of alpha7-nAChR function in hippocampal interneurons in culture shows that PMCA2 activity limits the duration of calcium elevations produced by the receptors. Unexpectedly, PMCA2 inhibition triggers rapid calcium-dependent loss of alpha7-nAChR clusters. This extreme regulatory response is mediated by CaMKII, involves proteasome activity, depends on the second intracellular loop of alpha7-nAChR subunits, and is specific in that it does not alter two other classes of calcium-permeable ionotropic receptors on the same neurons. A critical link is provided by the scaffold protein PSD-95 (postsynaptic density-95), which is associated with alpha7-nAChRs and constrains their mobility as revealed by single-particle tracking on neurons. The PSD-95 link is required for PMCA2-mediated removal of alpha7-nAChR clusters. This three-component combination of PMCA2, PSD-95, and alpha7-nAChR offers a novel mechanism for tight control of calcium dynamics in neurons.
ESTHER : Gomez-Varela_2012_J.Neurosci_32_6894
PubMedSearch : Gomez-Varela_2012_J.Neurosci_32_6894
PubMedID: 22593058

Title : Screens for active and stereoselective hydrolytic enzymes - Bottcher_2010_Methods.Mol.Biol_668_169
Author(s) : Bottcher D , Schmidt M , Bornscheuer UT
Ref : Methods Mol Biol , 668 :169 , 2010
Abstract : A procedure for the high-throughput screening (HTS) of esterases is described. This includes a pretest for discrimination of active and inactive clones using an agar plate overlay assay, the enzyme expression in microtiter plates and the measurement of activity and enantioselectivity (E) of the esterase variants using acetates of secondary alcohols as model substrates. Acetic acid released is converted in an enzyme cascade leading to the stoichiometric formation of NADH, which is quantified in a spectrophotometer. The method allows screening of several thousand mutants per day and has already been successfully applied to identify an esterase mutant with an E > 100 towards an important building block for organic synthesis. This protocol can also be used for lipases and possibly other hydrolases that are expressed in soluble form in conventional Escherichia coli strains.
ESTHER : Bottcher_2010_Methods.Mol.Biol_668_169
PubMedSearch : Bottcher_2010_Methods.Mol.Biol_668_169
PubMedID: 20830563

Title : Production of pig liver esterase in batch fermentation of E. coli Origami - Brusehaber_2010_Appl.Microbiol.Biotechnol_86_1337
Author(s) : Brusehaber E , Schwiebs A , Schmidt M , Bottcher D , Bornscheuer UT
Ref : Applied Microbiology & Biotechnology , 86 :1337 , 2010
Abstract : The establishment of a fermentation process for the production of pig liver esterase (PLE) in high yields is necessary for industrial applications. In our previous studies, we reported the recombinant expression of PLE in Escherichia coli Origami (DE3) in shake flask. Only a coexpression with chaperones GroEL/ES allowed the production of soluble and active enzyme. The optimization of the cultivation conditions, such as temperature, inducer concentrations, or media compositions to increase enzyme yield in a fermentation process is described here. Using fed-batch fermentation cell densities up to OD = 50 were obtained, but almost no active enzyme was expressed. Only batch fermentation was found suitable for production of active pig liver esterase and cell densities between OD = 7-13 and activities of 300-400 U L(-1) for isoenzyme PLE-1 (gammaPLE) and 1,400 U L(-1) for PLE-5 were obtained after 22 h total cultivation time or 18 h after induction of PLE expression, respectively.
ESTHER : Brusehaber_2010_Appl.Microbiol.Biotechnol_86_1337
PubMedSearch : Brusehaber_2010_Appl.Microbiol.Biotechnol_86_1337
PubMedID: 20024542

Title : Thermostable lipases from the extreme thermophilic anaerobic bacteria Thermoanaerobacter thermohydrosulfuricus SOL1 and Caldanaerobacter subterraneus subsp. tengcongensis - Royter_2009_Extremophiles_13_769
Author(s) : Royter M , Schmidt M , Elend C , Hobenreich H , Schafer T , Bornscheuer UT , Antranikian G
Ref : Extremophiles , 13 :769 , 2009
Abstract : Two novel genes encoding for heat and solvent stable lipases from strictly anaerobic extreme thermophilic bacteria Thermoanaerobacter thermohydrosulfuricus (LipTth) and Caldanaerobacter subterraneus subsp. tengcongensis (LipCst) were successfully cloned and expressed in E. coli. Recombinant proteins were purified to homogeneity by heat precipitation, hydrophobic interaction, and gel filtration chromatography. Unlike the enzymes from mesophile counterparts, enzymatic activity was measured at a broad temperature and pH range, between 40 and 90 degrees C and between pH 6.5 and 10; the half-life of the enzymes at 75 degrees C and pH 8.0 was 48 h. Inhibition was observed with 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride and phenylmethylsulfonylfluorid indicating that serine and thiol groups play a role in the active site of the enzymes. Gene sequence comparisons indicated very low identity to already described lipases from mesophilic and psychrophilic microorganisms. By optimal cultivation of E. coli Tuner (DE3) cells in 2-l bioreactors, a massive production of the recombinant lipases was achieved (53-2200 U/l) Unlike known lipases, the purified robust proteins are resistant against a large number of organic solvents (up to 99%) and detergents, and show activity toward a broad range of substrates, including triacylglycerols, monoacylglycerols, esters of secondary alcohols, and p-nitrophenyl esters. Furthermore, the enzyme from T. thermohydrosulfuricus is suitable for the production of optically pure compounds since it is highly S-stereoselective toward esters of secondary alcohols. The observed E values for but-3-yn-2-ol butyrate and but-3-yn-2-ol acetate of 21 and 16, respectively, make these enzymes ideal candidates for kinetic resolution of synthetically useful compounds.
ESTHER : Royter_2009_Extremophiles_13_769
PubMedSearch : Royter_2009_Extremophiles_13_769
PubMedID: 19579003
Gene_locus related to this paper: theet-q3ch51 , thete-TTE1809

Title : Protein engineering of carboxyl esterases by rational design and directed evolution - Schmidt_2009_Protein.Pept.Lett_16_1162
Author(s) : Schmidt M , Bottcher D , Bornscheuer UT
Ref : Protein Pept Lett , 16 :1162 , 2009
Abstract : In the past few years a considerable number of mutagenesis methods and high-throughput screening (HTS) systems have been developed and improved. In parallel, computer programs or software packages for molecular modeling have been further investigated. Thus, the number of examples for successful directed evolution and rational design is increasing constantly. In this review the essential mutagenesis methods and HTS systems, especially for esterases, are described and various examples for the application of these protein engineering tools are provided.
ESTHER : Schmidt_2009_Protein.Pept.Lett_16_1162
PubMedSearch : Schmidt_2009_Protein.Pept.Lett_16_1162
PubMedID: 19508186

Title : A versatile esterase from Bacillus subtilis: cloning, expression, characterization, and its application in biocatalysis - Schmidt_2007_Biotechnol.J_2_249
Author(s) : Schmidt M , Henke E , Heinze B , Kourist R , Hidalgo A , Bornscheuer UT
Ref : Biotechnol J , 2 :249 , 2007
Abstract : An esterase from Bacillus subtilis DSM402 (BS2) was cloned and functionally expressed in E. coli. The enzyme is active up to 50 degrees C, and the V(max) (1449 mM/min) and K(M) values (119 mM) were determined using p-nitrophenyl acetate as substrate. BS2 belongs to the few hydrolases that can act on tertiary alcohols and was therefore used to resolve racemic acetates of selected tertiary alcohols, but also to selectively remove the tert-butyl ester protecting group from peptides. In addition, the enzyme shows promiscuous amidase activity.
ESTHER : Schmidt_2007_Biotechnol.J_2_249
PubMedSearch : Schmidt_2007_Biotechnol.J_2_249
PubMedID: 17136743
Gene_locus related to this paper: bacsu-pnbae

Title : Regulator of G-protein signalling 3 redirects prototypical Gi-coupled receptors from Rac1 to RhoA activation - Vogt_2007_Cell.Signal_19_1229
Author(s) : Vogt A , Lutz S , Rumenapp U , Han L , Jakobs KH , Schmidt M , Wieland T
Ref : Cell Signal , 19 :1229 , 2007
Abstract : The small GTPases, Rac1 and RhoA, are pivotal regulators of several essential, but distinct cellular processes. Numerous G-protein-coupled receptors signal to these GTPases, but with different specificities. Specifically, Gi-coupled receptors (GiPCRs) are generally believed to activate Rac1, but not RhoA, a process involving Gbetagamma-dimers and phosphatidylinositol 3-kinase (PI3K). Here we show that, depending on the expression level of the 519 amino acid isoform of regulator of G-protein signalling 3 (RGS3L), prototypical GiPCRs, like M2 muscarinic, A1 adenosine, and alpha2-adrenergic receptors, activate either Rac1 or RhoA in human embryonic kidney cells and neonatal rat cardiomyocyte-derived H10 cells. The switch from Rac1 to RhoA activation in H10 cells was controlled by fibroblast growth factor-2 (FGF-2), lowering the expression of RGS3L. Activation of both, Rac1 and RhoA, seen at low and high expression levels of RGS3L, respectively, was sensitive to pertussis toxin and the PI3K inhibitor LY294002 and mediated by Gbetagamma-dimers. We conclude that RGS3L functions as a molecular switch, redirecting GiPCRs via Gbetagamma-dimers and PI3K from Rac1 to RhoA activation. Considering the essential roles of Rac1 and RhoA in many signalling pathways, this additional function of RGS3L indicates a specific role of this protein in cellular signalling networks.
ESTHER : Vogt_2007_Cell.Signal_19_1229
PubMedSearch : Vogt_2007_Cell.Signal_19_1229
PubMedID: 17300916

Title : Genomic sequence of chorioallantois vaccinia virus Ankara, the ancestor of modified vaccinia virus Ankara - Meisinger-Henschel_2007_J.Gen.Virol_88_3249
Author(s) : Meisinger-Henschel C , Schmidt M , Lukassen S , Linke B , Krause L , Konietzny S , Goesmann A , Howley P , Chaplin P , Suter M , Hausmann J
Ref : Journal of General Virology , 88 :3249 , 2007
Abstract : Chorioallantois vaccinia virus Ankara (CVA) is the parental virus of modified vaccinia virus Ankara (MVA), which was derived from CVA by more than 570 passages in chicken embryo fibroblasts (CEF). MVA became severely host-cell-restricted to avian cells and has strongly diminished virulence in mammalian hosts, while maintaining good immunogenicity. We determined the complete coding sequence of the parental CVA and mapped the exact positions of the six major deletions that emerged in the MVA genome. All six major deletions occurred in regions of the CVA genome where one or more truncated or fragmented open reading frames (ORFs) pre-existed. The CVA genome contains 229 ORFs of which 51 are fragments of full-length orthopoxvirus (OPV) genes, including fragmented orthologues of C9L and M1L (encoding two well-conserved ankyrin-like proteins), A39R (encoding a semaphorin-like protein) and A55R (encoding a kelch-like protein). Phylogenetic analysis demonstrated that MVA was most closely related to CVA, followed by the vaccinia virus (VACV) strain DUKE, a patient-derived isolate of the Dryvax vaccine virus. Loss or mutation of genes outside the six major deletions are assumed to contribute to the restricted host range phenotype of MVA. In support of this notion, deletions, insertions and non-synonymous mutations were found in 122 of the 195 ORFs remaining in MVA when compared with their CVA counterparts. Thus, detailed knowledge of the CVA genomic sequence is a prerequisite to further dissect the genetic basis of the MVA host range phenotype as well as the particular immunological properties of MVA.
ESTHER : Meisinger-Henschel_2007_J.Gen.Virol_88_3249
PubMedSearch : Meisinger-Henschel_2007_J.Gen.Virol_88_3249
PubMedID: 18024893
Gene_locus related to this paper: cowvi-M5L

Title : Direct stimulation of receptor-controlled phospholipase D1 by phospho-cofilin - Han_2007_EMBO.J_26_4189
Author(s) : Han L , Stope MB , de Jesus ML , Oude Weernink PA , Urban M , Wieland T , Rosskopf D , Mizuno K , Jakobs KH , Schmidt M
Ref : EMBO Journal , 26 :4189 , 2007
Abstract : The activity state of cofilin, which controls actin dynamics, is driven by a phosphorylation-dephosphorylation cycle. Phosphorylation of cofilin by LIM-kinases results in its inactivation, a process supported by 14-3-3zeta and reversed by dephosphorylation by slingshot phosphatases. Here we report on a novel cellular function for the phosphorylation-dephosphorylation cycle of cofilin. We demonstrate that muscarinic receptor-mediated stimulation of phospholipase D1 (PLD1) is controlled by LIM-kinase, slingshot phosphatase as well as 14-3-3zeta, and requires phosphorylatable cofilin. Cofilin directly and specifically interacts with PLD1 and upon phosphorylation by LIM-kinase1, stimulates PLD1 activity, an effect mimicked by phosphorylation-mimic cofilin mutants. The interaction of cofilin with PLD1 is under receptor control and encompasses a PLD1-specific fragment (aa 585-712). Expression of this fragment suppresses receptor-induced cofilin-PLD1 interaction as well as PLD stimulation and actin stress fiber formation. These data indicate that till now designated inactive phospho-cofilin exhibits an active cellular function, and suggest that phospho-cofilin by its stimulatory effect on PLD1 may control a large variety of cellular functions.
ESTHER : Han_2007_EMBO.J_26_4189
PubMedSearch : Han_2007_EMBO.J_26_4189
PubMedID: 17853892

Title : Directed evolution of an esterase from Pseudomonas fluorescens yields a mutant with excellent enantioselectivity and activity for the kinetic resolution of a chiral building block - Schmidt_2006_Chembiochem_7_805
Author(s) : Schmidt M , Hasenpusch D , Kahler M , Kirchner U , Wiggenhorn K , Langel W , Bornscheuer UT
Ref : Chembiochem , 7 :805 , 2006
Abstract : A triple mutant of an esterase from Pseudomonas fluorescens (PFE) that was created by directed evolution exhibited high enantioselectivity (E=89) in a kinetic resolution and yielded the building block (S)-but-3-yn-2-ol. Surprisingly, a mutation close to the active site caused the formation of inclusion bodies, but remote mutations were found to be responsible for the high selectivity. Back mutations gave a variant (double mutant PFE Ile76Val/Val175Ala) that showed excellent selectivity (E=96) and activity (20 min for 50% conversion, which corresponds to 1.25 U per mg of protein).
ESTHER : Schmidt_2006_Chembiochem_7_805
PubMedSearch : Schmidt_2006_Chembiochem_7_805
PubMedID: 16575940

Title : Cyclic AMP-dependent and Epac-mediated activation of R-Ras by G protein-coupled receptors leads to phospholipase D stimulation - Lopez_2006_J.Biol.Chem_281_21837
Author(s) : Lopez De Jesus M , Stope MB , Oude Weernink PA , Mahlke Y , Borgermann C , Ananaba VN , Rimmbach C , Rosskopf D , Michel MC , Jakobs KH , Schmidt M
Ref : Journal of Biological Chemistry , 281 :21837 , 2006
Abstract : The activation of the Ras-related GTPase R-Ras, which has been implicated in the regulation of various cellular functions, by G protein-coupled receptors (GPCRs) was studied in HEK-293 cells stably expressing the M3 muscarinic acetylcholine receptor (mAChR), which can couple to several types of heterotrimeric G proteins. Activation of the receptor induced a very rapid and transient activation of R-Ras. Studies with inhibitors and activators of various signaling pathways indicated that R-Ras activation by the M3 mAChR is dependent on cyclic AMP formation but is independent of protein kinase A. Similar to the rather promiscuous M3 mAChR, two typical G(s)-coupled receptors also induced R-Ras activation. The receptor actions were mimicked by an Epac-specific cyclic AMP analog and suppressed by depletion of endogenous Epac1 by small interfering RNAs, as well as expression of a cyclic AMP binding-deficient Epac1 mutant, but not by expression of dominant negative Rap GTPases. In vitro studies demonstrated that Epac1 directly interacts with R-Ras and catalyzes GDP/GTP exchange at this GTPase. Finally, it is shown that the cyclic AMP- and Epac-activated R-Ras plays a major role in the M3 mAChR-mediated stimulation of phospholipase D but not phospholipase C. Collectively, our data indicate that GPCRs rapidly activate R-Ras, that R-Ras activation by the GPCRs is apparently directly induced by cyclic AMP-regulated Epac proteins, and that activated R-Ras specifically controls GPCR-mediated phospholipase D stimulation.
ESTHER : Lopez_2006_J.Biol.Chem_281_21837
PubMedSearch : Lopez_2006_J.Biol.Chem_281_21837
PubMedID: 16754664

Title : High-throughput assays for lipases and esterases - Schmidt_2005_Biomol.Eng_22_51
Author(s) : Schmidt M , Bornscheuer UT
Ref : Biomol Eng , 22 :51 , 2005
Abstract : In the past few years a considerable number of high-throughput screening (HTS) systems have been developed, especially for lipases and esterases. In this review, a range of HTS methods for the directed evolution of these hydrolases are covered. This includes spectrophotometric and fluorimetric formats as well as other approaches to allow for fast, efficient and reliable identification of desired enzyme variants within large mutant libraries. In addition, methods for library creation and application of lipases and esterases are briefly covered.
ESTHER : Schmidt_2005_Biomol.Eng_22_51
PubMedSearch : Schmidt_2005_Biomol.Eng_22_51
PubMedID: 15857783

Title : Enzymatic removal of carboxyl protecting groups. 1. Cleavage of the tert-butyl moiety - Schmidt_2005_J.Org.Chem_70_3737
Author(s) : Schmidt M , Barbayianni E , Fotakopoulou I , Hohne M , Constantinou-Kokotou V , Bornscheuer UT , Kokotos G
Ref : J Org Chem , 70 :3737 , 2005
Abstract : [reaction: see text] A recent discovery that a certain amino acid motif (GGG(A)X-motif) in lipases and esterases determines their activity toward tertiary alcohols prompted us to investigate the use of these biocatalysts in the mild and selective removal of tert-butyl protecting groups in amino acid derivatives and related compounds. An esterase from Bacillus subtilis (BsubpNBE) and lipase A from Candida antarctica (CAL-A) were identified as the most active enzymes, which hydrolyzed a range of tert-butyl esters of protected amino acids (e.g., Boc-Tyr-O(t)Bu, Z-GABA-O(t)Bu, Fmoc-GABA-O(t)Bu) in good to high yields and left Boc, Z, and Fmoc-protecting groups intact.
ESTHER : Schmidt_2005_J.Org.Chem_70_3737
PubMedSearch : Schmidt_2005_J.Org.Chem_70_3737
PubMedID: 15845019
Gene_locus related to this paper: canan-lipasA

Title : Sol i 1, the phospholipase allergen of imported fire ant venom - Hoffman_2005_J.Allergy.Clin.Immunol_115_611
Author(s) : Hoffman DR , Sakell RH , Schmidt M
Ref : J Allergy Clin Immunol , 115 :611 , 2005
Abstract : BACKGROUND: Sol i 1, the venom phospholipase of imported fire ant venom is an important allergen and exhibits some cross-reactivity with IgE antibodies from patients sensitized to other Hymenoptera venoms. OBJECTIVE: To determine the primary structure of Sol i 1 and evaluate the roles of protein and carbohydrate epitopes in its cross-reactivity.
METHODS: Sol i 1 was purified from venom, proteolytic peptides prepared and amino acid sequences obtained. The cDNA for Sol i 1 was cloned, sequenced, and compared with sequences of other wasp venom phospholipases. The role of carbohydrate epitopes in the cross-reactivity with other Hymenoptera venoms was studied by RAST inhibition.
RESULTS: The sequence identified Sol i 1 as a lipase of the GX class, lipoprotein lipase superfamily, pancreatic lipase homologous family and RP2 subgroup phospholipases as are the vespid venom phospholipases. The 148 residues identified by amino acid sequencing represent about 48% of the translated cDNA sequence. Sol i 1 was 31-32% identical to yellow jacket phospholipases. The identical regions of sequence were clustered in the domain which forms the serine hydrolase active site. Mannosylated N-glycans could completely inhibit binding of IgE from honeybee venom sensitized patients to Sol i 1. Inhibition by glycan of IgE binding from yellow jacket venom sensitized patients was low or absent for three of eight sera and substantial, but not complete for five sera.
CONCLUSIONS: Sol i 1 is related to wasp venom phospholipases. Cross-reactivity with honeybee venom is caused by carbohydrate, whereas cross-reactivity with yellow jacket venom involves reactivity with both carbohydrate determinants of hyaluronidase and high molecular weight proteins and phospholipase protein determinants.
ESTHER : Hoffman_2005_J.Allergy.Clin.Immunol_115_611
PubMedSearch : Hoffman_2005_J.Allergy.Clin.Immunol_115_611
PubMedID: 15753912
Gene_locus related to this paper: solin-q68kk0 , vessq-pa1

Title : Enzymatic removal of carboxyl protecting groups. 2. Cleavage of the benzyl and methyl moieties - Barbayianni_2005_J.Org.Chem_70_8730
Author(s) : Barbayianni E , Fotakopoulou I , Schmidt M , Constantinou-Kokotou V , Bornscheuer UT , Kokotos G
Ref : J Org Chem , 70 :8730 , 2005
Abstract : [reaction: see text] Enzymes are versatile reagents for the efficient removal of methyl and benzyl protecting groups. An esterase from Bacillus subtilis (BS2) and a lipase from Candida antarctica (CAL-A) allow a mild and selective removal of these moieties in high yields without affecting other functional groups.
ESTHER : Barbayianni_2005_J.Org.Chem_70_8730
PubMedSearch : Barbayianni_2005_J.Org.Chem_70_8730
PubMedID: 16238302

Title : Directed evolution of lipases and esterases -
Author(s) : Schmidt M , Baumann M , Henke E , Konarzycka-Bessler M , Bornscheuer UT
Ref : Methods Enzymol , 388 :199 , 2004
PubMedID: 15289073

Title : Inhibition of phospholipase C-epsilon by Gi-coupled receptors - vom_2004_Cell.Signal_16_921
Author(s) : vom Dorp F , Sari AY , Sanders H , Keiper M , Oude Weernink PA , Jakobs KH , Schmidt M
Ref : Cell Signal , 16 :921 , 2004
Abstract : We recently reported that several Gs-coupled receptors stimulate phospholipase C (PLC)-epsilon via increased formation of cyclic AMP and subsequent activation of the small GTPase Rap2B by the cyclic AMP-activated exchange factor Epac1. Here we show by studies in HEK-293 and N1E-115 neuroblastoma cells that this stimulation induced by Gs-coupled receptors or the direct adenylyl cyclase activator, forskolin, is potently inhibited by Gi-coupled receptors, known to inhibit cyclic AMP formation. PLC inhibition by the overexpressed M2 muscarinic receptor and the endogenously expressed sphingosine-1-phosphate and delta-opioid receptors was fully pertussis toxin-sensitive and accompanied by a reduction in Rap2B activation induced by Gs-coupled receptors. In contrast, Rap2B activation and PLC stimulation induced by membrane-permeable cyclic AMP analogues, including an Epac-specific activator, or PLC stimulation caused by constitutively active Rap2B were not affected by the Gi-coupled receptors. In summary, our data indicate that Gi-coupled receptors can inhibit PLC-epsilon, most likely by suppressing formation of cyclic AMP required for Epac-mediated Rap2B activation.
ESTHER : vom_2004_Cell.Signal_16_921
PubMedSearch : vom_2004_Cell.Signal_16_921
PubMedID: 15157671

Title : Stimulation of phospholipase C-epsilon by the M3 muscarinic acetylcholine receptor mediated by cyclic AMP and the GTPase Rap2B - Evellin_2002_J.Biol.Chem_277_16805
Author(s) : Evellin S , Nolte J , Tysack K , vom Dorp F , Thiel M , Weernink PA , Jakobs KH , Webb EJ , Lomasney JW , Schmidt M
Ref : Journal of Biological Chemistry , 277 :16805 , 2002
Abstract : Stimulation of phospholipase C (PLC) by G(q)-coupled receptors such as the M(3) muscarinic acetylcholine receptor (mAChR) is caused by direct activation of PLC-beta enzymes by Galpha(q) proteins. We have recently shown that G(s)-coupled receptors can stimulate PLC-epsilon, apparently via formation of cyclic AMP and activation of the Ras-related GTPase Rap2B. Here we report that PLC stimulation by the M(3) mAChR expressed in HEK-293 cells also involves, in part, similar mechanisms. M(3) mAChR-mediated PLC stimulation and [Ca(2+)](i) increase were reduced by 2',5'-dideoxyadenosine (dd-Ado), a direct adenylyl cyclase inhibitor. On the other hand, overexpression of Galpha(s) or Epac1, a cyclic AMP-regulated guanine nucleotide exchange factor for Rap GTPases, enhanced M(3) mAChR-mediated PLC stimulation. Inactivation of Ras-related GTPases with clostridial toxins suppressed the M(3) mAChR responses. The inhibitory toxin effects were mimicked by expression of inactive Rap2B, but not of other inactive GTPases (Rac1, Ras, RalA, Rap1A, and Rap2A). Activation of the M(3) mAChR induced GTP loading of Rap2B, an effect strongly enhanced by overexpression of Galpha(s) and inhibited by dd-Ado. Overexpression of PLC-epsilon and PLC-beta1, but not PLC-gamma1 or PLC-delta1, enhanced M(3) mAChR-mediated PLC stimulation and [Ca(2+)](i) increase. In contrast, expression of a catalytically inactive PLC-epsilon mutant reduced PLC stimulation by the M(3) mAChR and abrogated the potentiating effect of Galpha(s). In conclusion, our findings suggest that PLC stimulation by the M(3) mAChR is a composite action of PLC-beta1 stimulation by Galpha(q) and stimulation of PLC-epsilon apparently mediated by G(s)-dependent cyclic AMP formation and subsequent activation of Rap2B.
ESTHER : Evellin_2002_J.Biol.Chem_277_16805
PubMedSearch : Evellin_2002_J.Biol.Chem_277_16805
PubMedID: 11877431

Title : The M3 muscarinic acetylcholine receptor expressed in HEK-293 cells signals to phospholipase D via G12 but not Gq-type G proteins: regulators of G proteins as tools to dissect pertussis toxin-resistant G proteins in receptor-effector coupling - Rumenapp_2001_J.Biol.Chem_276_2474
Author(s) : Rumenapp U , Asmus M , Schablowski H , Woznicki M , Han L , Jakobs KH , Fahimi-Vahid M , Michalek C , Wieland T , Schmidt M
Ref : Journal of Biological Chemistry , 276 :2474 , 2001
Abstract : The M(3) muscarinic acetylcholine receptor (mAChR) expressed in HEK-293 cells couples to G(q) and G(12) proteins and stimulates phospholipase C (PLC) and phospholipase D (PLD) in a pertussis toxin-insensitive manner. To determine the type of G protein mediating M(3) mAChR-PLD coupling in comparison to M(3) mAChR-PLC coupling, we expressed various Galpha proteins and regulators of the G protein signaling (RGS), which act as GTPase-activating proteins for G(q)- or G(12)-type G proteins. PLD stimulation by the M(3) mAChR was enhanced by the overexpression of Galpha(12) and Galpha(13), whereas the overexpression of Galpha(q) strongly increased PLC activity without affecting PLD activity. Expression of the RGS homology domain of Lsc, which acts specifically on Galpha(12) and Galpha(13), blunted the M(3) mAChR-induced PLD stimulation without affecting PLC stimulation. On the other hand, overexpression of RGS4, which acts on Galpha(q)- but not Galpha(12)-type G proteins, suppressed the M(3) mAChR-induced PLC stimulation without altering PLD stimulation. We conclude that the M(3) mAChR in HEK-293 cells apparently signals to PLD via G(12)- but not G(q)-type G proteins and that G protein subtype-selective RGS proteins can be used as powerful tools to dissect the pertussis toxin-resistant G proteins and their role in receptor-effector coupling.
ESTHER : Rumenapp_2001_J.Biol.Chem_276_2474
PubMedSearch : Rumenapp_2001_J.Biol.Chem_276_2474
PubMedID: 11036069

Title : G protein-coupled receptor-induced sensitization of phospholipase C stimulation by receptor tyrosine kinases - Schmidt_2000_J.Biol.Chem_275_32603
Author(s) : Schmidt M , Frings M , Mono ML , Guo Y , Weernink PA , Evellin S , Han L , Jakobs KH
Ref : Journal of Biological Chemistry , 275 :32603 , 2000
Abstract : Activation of stably expressed M(2) and M(3) muscarinic acetylcholine receptors (mAChRs) as well as of endogenously expressed lysophosphatidic acid and purinergic receptors in HEK-293 cells can induce a long lasting potentiation of phospholipase C (PLC) stimulation by these and other G protein-coupled receptors (GPCRs). Here, we report that GPCRs can induce an up-regulation of PLC stimulation by receptor tyrosine kinases (RTKs) as well and provide essential mechanistic characteristics of this sensitization process. Pretreatment of HEK-293 cells for 2 min with carbachol, a mAChR agonist, lysophosphatidic acid, or ATP, followed by agonist washout, strongly increased (by 2-3-fold) maximal PLC stimulation (measured >/=40 min later) by epidermal growth factor and platelet-derived growth factor, but not insulin, and largely enhanced PLC sensitivity to these RTK agonists. The up-regulation of RTK-induced PLC stimulation was cycloheximide-insensitive and was observed for up to approximately 90 min after removal of the GPCR agonist. Sensitization of receptor-induced PLC stimulation caused by prior M(2) mAChR activation was fully prevented by pertussis toxin and strongly reduced by expression of Gbetagamma scavengers. Furthermore, inhibition of conventional protein kinase C (PKC) isoenzymes and chelation of intracellular Ca(2+) suppressed the sensitization process, while overexpression of PKC-alpha, but not PKC-betaI, further enhanced the M(2) mAChR-induced sensitization of PLC stimulation. None of these treatments affected acute PLC stimulation by either GPCR or RTK agonists. Taken together, short term activation of GPCRs can induce a strong and long lasting sensitization of PLC stimulation by RTKs, a process apparently involving G(i)-derived Gbetagammas as well as increases in intracellular Ca(2+) and activation of a PKC isoenzyme, most likely PKC-alpha.
ESTHER : Schmidt_2000_J.Biol.Chem_275_32603
PubMedSearch : Schmidt_2000_J.Biol.Chem_275_32603
PubMedID: 10908568

Title : The mycosubtilin synthetase of Bacillus subtilis ATCC6633: a multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase - Duitman_1999_Proc.Natl.Acad.Sci.U.S.A_96_13294
Author(s) : Duitman EH , Hamoen LW , Rembold M , Venema G , Seitz H , Saenger W , Bernhard F , Reinhardt R , Schmidt M , Ullrich C , Stein T , Leenders F , Vater J
Ref : Proc Natl Acad Sci U S A , 96 :13294 , 1999
Abstract : Bacillus subtilis strain ATCC6633 has been identified as a producer of mycosubtilin, a potent antifungal peptide antibiotic. Mycosubtilin, which belongs to the iturin family of lipopeptide antibiotics, is characterized by a beta-amino fatty acid moiety linked to the circular heptapeptide Asn-Tyr-Asn-Gln-Pro-Ser-Asn, with the second, third, and sixth position present in the D-configuration. The gene cluster from B. subtilis ATCC6633 specifying the biosynthesis of mycosubtilin was identified. The putative operon spans 38 kb and consists of four ORFs, designated fenF, mycA, mycB, and mycC, with strong homologies to the family of peptide synthetases. Biochemical characterization showed that MycB specifically adenylates tyrosine, as expected for mycosubtilin synthetase, and insertional mutagenesis of the operon resulted in a mycosubtilin-negative phenotype. The mycosubtilin synthetase reveals features unique for peptide synthetases as well as for fatty acid synthases: (i) The mycosubtilin synthase subunit A (MycA) combines functional domains derived from peptide synthetases, amino transferases, and fatty acid synthases. MycA represents the first example of a natural hybrid between these enzyme families. (ii) The organization of the synthetase subunits deviates from that commonly found in peptide synthetases. On the basis of the described characteristics of the mycosubtilin synthetase, we present a model for the biosynthesis of iturin lipopeptide antibiotics. Comparison of the sequences flanking the mycosubtilin operon of B. subtilis ATCC6633, with the complete genome sequence of B. subtilis strain 168 indicates that the fengycin and mycosubtilin lipopeptide synthetase operons are exchanged between the two B. subtilis strains.
ESTHER : Duitman_1999_Proc.Natl.Acad.Sci.U.S.A_96_13294
PubMedSearch : Duitman_1999_Proc.Natl.Acad.Sci.U.S.A_96_13294
PubMedID: 10557314
Gene_locus related to this paper: bacsu-MYCC

Title : Role of sphingosine kinase in Ca(2+) signalling by epidermal growth factor receptor - Meyer_1999_FEBS.Lett_461_217
Author(s) : Meyer zu Heringdorf D , Lass H , Kuchar I , Alemany R , Guo Y , Schmidt M , Jakobs KH
Ref : FEBS Letters , 461 :217 , 1999
Abstract : Contribution of sphingosine kinase (SPK)-catalyzed production of sphingosine-1-phosphate (SPP), in comparison to phospholipase C (PLC), to Ca(2+) signalling by epidermal growth factor (EGF) was studied in two HEK-293 cell clones (HEK2 and HEK3), expressing functional EGF receptors and exhibiting release of stored Ca(2+) by intracellular SPP. In HEK3 cells, EGF increased [Ca(2+)](i) and stimulated both, SPK and PLC. [Ca(2+)](i) increase, but not PLC stimulation, was strongly reduced by SPK inhibition. In HEK2 cells, EGF similarly stimulated PLC, but did not increase [Ca(2+)](i) or stimulate SPK, suggesting that intracellular SPP production plays a major role for Ca(2+) signalling by EGF in HEK-293 cells.
ESTHER : Meyer_1999_FEBS.Lett_461_217
PubMedSearch : Meyer_1999_FEBS.Lett_461_217
PubMedID: 10567700

Title : A role for rho-kinase in rho-controlled phospholipase D stimulation by the m3 muscarinic acetylcholine receptor - Schmidt_1999_J.Biol.Chem_274_14648
Author(s) : Schmidt M , Voss M , Weernink PA , Wetzel J , Amano M , Kaibuchi K , Jakobs KH
Ref : Journal of Biological Chemistry , 274 :14648 , 1999
Abstract : Stimulation of phospholipase D (PLD) by membrane receptors is now recognized as a major signal transduction pathway involved in diverse cellular functions. Rho proteins control receptor signaling to PLD, and these GTPases have been shown to directly stimulate purified recombinant PLD1 enzymes in vitro. Here we report that stimulation of PLD activity, measured in the presence of phosphatidylinositol 4,5-bisphosphate, by RhoA in membranes of HEK-293 cells expressing the m3 muscarinic acetylcholine receptor (mAChR) is phosphorylation-dependent. Therefore, the possible involvement of the RhoA-stimulated serine/threonine kinase, Rho-kinase, was investigated. Overexpression of Rho-kinase and constitutively active Rho-kinase (Rho-kinase-CAT) but not of kinase-deficient Rho-kinase-CAT markedly increased m3 mAChR-mediated but not protein kinase C-mediated PLD stimulation, similar to overexpression of RhoA. Expression of the Rho-inactivating C3 transferase abrogated the stimulatory effect of wild-type Rho-kinase, but not of Rho-kinase-CAT. Recombinant Rho-kinase-CAT mimicked the phosphorylation-dependent PLD stimulation by RhoA in HEK-293 cell membranes. Finally, the Rho-kinase inhibitor HA-1077 largely inhibited RhoA-induced PLD stimulation in membranes as well as PLD stimulation by the m3 mAChR but not by protein kinase C in intact HEK-293 cells. We conclude that Rho-kinase is involved in Rho-dependent PLD stimulation by the G protein-coupled m3 mAChR in HEK-293 cells. Thus, our findings identify Rho-kinase as a novel player in the receptor-controlled PLD signaling pathway.
ESTHER : Schmidt_1999_J.Biol.Chem_274_14648
PubMedSearch : Schmidt_1999_J.Biol.Chem_274_14648
PubMedID: 10329658

Title : The ADP-ribosylation factor (ARF)-related GTPase ARF-related protein binds to the ARF-specific guanine nucleotide exchange factor cytohesin and inhibits the ARF-dependent activation of phospholipase D - Schurmann_1999_J.Biol.Chem_274_9744
Author(s) : Schurmann A , Schmidt M , Asmus M , Bayer S , Fliegert F , Koling S , Massmann S , Schilf C , Subauste MC , Voss M , Jakobs KH , Joost HG
Ref : Journal of Biological Chemistry , 274 :9744 , 1999
Abstract : ADP-ribosylation factor-related protein (ARP) is a membrane-associated GTPase with remote similarity to the family of ADP-ribosylation factors (ARF). In a yeast two-hybrid screen designed to identify proteins interacting with ARP, we isolated a partial cDNA of the ARF-specific guanine nucleotide exchange factor mSec7-1/cytohesin encoding its N terminus and most of the Sec7 domain (codons 1-200). ARP and ARP-Q79L (GTPase-negative ARP) exhibited a higher affinity to mSec7-1-(1-200) than ARP-T31N (nucleotide exchange-defective ARP) in the two-hybrid assay. Similarly, full-length [35S]mSec7-1/cytohesin was specifically adsorbed to glutathione-Sepharose loaded with glutathione S-transferase (GST)-ARP-Q79L, GST-ARP, or GST-ARP-T31N, the latter exhibiting the lowest binding affinity. Overexpression of ARP-Q79L, but not of ARP-T31N, in COS-7 cells reduced the fluorescence from co-expressed green fluorescent protein fused with mSec7-1/cytohesin or mSec7-2/ARNO in plasma membranes as detected by deconvolution microscopy. Recombinant ARP and ARP-Q79L, but not ARP-T31N, inhibited the phospholipase D (PLD) activity stimulated by mSec7-2/ARNO and ARF in a system of isolated membranes. Furthermore, transfection of HEK-293 cells with ARP or ARP-Q79L, but not ARP-T31N, inhibited the muscarinic acetylcholine receptor-3 induced PLD stimulation and translocation of ARF from cytosol to membranes. These data suggest that the GTP-bound form of ARP specifically binds mSec7-1/cytohesin, and that ARP may be involved in a pathway inhibiting the ARF-controlled activity of PLD.
ESTHER : Schurmann_1999_J.Biol.Chem_274_9744
PubMedSearch : Schurmann_1999_J.Biol.Chem_274_9744
PubMedID: 10092663

Title : RhoA-sensitive trafficking of muscarinic acetylcholine receptors - Vogler_1999_J.Pharmacol.Exp.Ther_288_36
Author(s) : Vogler O , Krummenerl P , Schmidt M , Jakobs KH , van Koppen CJ
Ref : Journal of Pharmacology & Experimental Therapeutics , 288 :36 , 1999
Abstract : The clathrin-mediated sequestration pathway is used by non-G protein-coupled receptors (e.g., transferrin receptors) and a large number of G protein-coupled receptors, including beta-2 adrenoceptors and various muscarinic acetylcholine receptor (mAChR) subtypes. Recently, the ubiquitously expressed small GTPase RhoA has been implicated as a negative regulator of transferrin receptor internalization. Because mAChRs and other G protein-coupled receptors are able to activate RhoA, we investigated in HEK-293 cells whether RhoA regulates the sequestration of m1 and m2 mAChRs, which internalize via clathrin-coated and nonclathrin-coated vesicles in HEK-293 cells, respectively. Overexpression of wild-type RhoA inhibited agonist-induced sequestration of both m1 and m2 mAChRs by as much as 70%. Inhibition could be reversed by coexpression of Clostridium botulinum C3 transferase, which inactivates RhoA by ADP-ribosylation. Overexpression of C3 transferase alone had no effect on m1 and m2 mAChR sequestration. In addition, overexpression of RhoA inhibited m1 and m2 mAChR transport to the plasma membrane by 60 and 31%, respectively, which was blocked by coexpression of C3 transferase. We conclude that RhoA is not an endogenous regulator of mAChR sequestration, but when overexpressed, strongly inhibits mAChR trafficking (i.e., sequestration and transport to the plasma membrane) in HEK-293 cells.
ESTHER : Vogler_1999_J.Pharmacol.Exp.Ther_288_36
PubMedSearch : Vogler_1999_J.Pharmacol.Exp.Ther_288_36
PubMedID: 9862750

Title : Regulation of muscarinic acetylcholine receptor sequestration and function by beta-arrestin - Vogler_1999_J.Biol.Chem_274_12333
Author(s) : Vogler O , Nolte B , Voss M , Schmidt M , Jakobs KH , van Koppen CJ
Ref : Journal of Biological Chemistry , 274 :12333 , 1999
Abstract : After activation, agonist-occupied G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases and bind cytosolic beta-arrestins, which uncouple the receptors from their cognate G proteins. Recent studies on the beta2-adrenergic receptor have demonstrated that beta-arrestin also targets the receptors to clathrin-coated pits for subsequent internalization and activation of mitogen-activated protein kinases. We and others have previously shown that muscarinic acetylcholine receptors (mAChRs) of the m1, m3, and m4 subtype require functional dynamin to sequester into HEK-293 tsA201 cells, whereas m2 mAChRs sequester in a dynamin-independent manner. To investigate the role of beta-arrestin in mAChR sequestration, we determined the effect of overexpressing beta-arrestin-1 and the dominant-negative inhibitor of beta-arrestin-mediated receptor sequestration, beta-arrestin-1 V53D, on mAChR sequestration and function. Sequestration of m1, m3, and m4 mAChRs was suppressed by 60-75% in cells overexpressing beta-arrestin-1 V53D, whereas m2 mAChR sequestration was affected by less than 10%. In addition, overexpression of beta-arrestin-1 V53D as well as dynamin K44A significantly suppressed m1 mAChR-mediated activation of mitogen-activated protein kinases. Finally, we investigated whether mAChRs sequester into clathrin-coated vesicles by overexpressing Hub, a dominant-negative clathrin mutant. Although sequestration of m1, m3, and m4 mAChRs was inhibited by 50-70%, m2 mAChR sequestration was suppressed by less than 10%. We conclude that m1, m3, and m4 mAChRs expressed in HEK-293 tsA201 cells sequester into clathrin-coated vesicles in a beta-arrestin- and dynamin-dependent manner, whereas sequestration of m2 mAChRs in these cells is largely independent of these proteins.
ESTHER : Vogler_1999_J.Biol.Chem_274_12333
PubMedSearch : Vogler_1999_J.Biol.Chem_274_12333
PubMedID: 10212203

Title : Phospholipase D stimulation by receptor tyrosine kinases mediated by protein kinase C and a Ras\/Ral signaling cascade - Voss_1999_J.Biol.Chem_274_34691
Author(s) : Voss M , Weernink PA , Haupenthal S , Moller U , Cool RH , Bauer B , Camonis JH , Jakobs KH , Schmidt M
Ref : Journal of Biological Chemistry , 274 :34691 , 1999
Abstract : Stimulation of phospholipase D (PLD) in HEK-293 cells expressing the M(3) muscarinic receptor by phorbol ester-activated protein kinase C (PKC) apparently involves Ral GTPases. We report here that PKC, but not muscarinic receptor-induced PLD stimulation in these cells, is strongly and specifically reduced by expression of dominant-negative RalA, G26A RalA, as well as dominant-negative Ras, S17N Ras. In contrast, overexpression of the Ras-activated Ral-specific guanine nucleotide exchange factor, Ral-GDS, specifically enhanced PKC-induced PLD stimulation. Moreover, recombinant Ral-GDS potentiated Ral-dependent PKC-induced PLD stimulation in membranes. Epidermal growth factor, platelet-derived growth factor, and insulin, ligands for receptor tyrosine kinases (RTKs) endogenously expressed in HEK-293 cells, apparently use the PKC- and Ras/Ral-dependent pathway for PLD stimulation. First, PLD stimulation by the RTK agonists was prevented by PKC inhibition and PKC down-regulation. Second, expression of dominant-negative RalA and Ras mutants strongly reduced RTK-induced PLD stimulation. Third, overexpression of Ral-GDS largely potentiated PLD stimulation by the RTK agonists. Finally, using the Ral binding domain of the Ral effector RLIP as an activation-specific probe for Ral proteins, it is demonstrated that endogenous RalA is activated by phorbol ester and RTK agonists. Taken together, strong evidence is provided that RTK-induced PLD stimulation in HEK-293 cells is mediated by PKC and a Ras/Ral signaling cascade.
ESTHER : Voss_1999_J.Biol.Chem_274_34691
PubMedSearch : Voss_1999_J.Biol.Chem_274_34691
PubMedID: 10574935

Title : Tyrosine-phosphorylation-dependent and rho-protein-mediated control of cellular phosphatidylinositol 4,5-bisphosphate levels - Rumenapp_1998_Biochem.J_334 ( Pt 3)_625
Author(s) : Rumenapp U , Schmidt M , Olesch S , Ott S , Eichel-Streiber CV , Jakobs KH
Ref : Biochemical Journal , 334 ( Pt 3) :625 , 1998
Abstract : The polyphosphoinositide PtdIns(4,5)P2, best known as a substrate for phospholipase C isozymes, has recently been recognized to be involved in a variety of other cellular processes. The aim of this study was to examine whether the cellular levels of this versatile phospholipid are controlled by tyrosine phosphorylation. The studies were performed in human embryonic kidney (HEK)-293 cells stably expressing the M3 muscarinic acetylcholine receptor. Inhibition of tyrosine phosphatases by pervanadate induced an up-to-approx.-2. 5-fold increase in the total cellular level of PtdIns(4,5)P2, which was both time- and concentration-dependent. In contrast, the tyrosine kinase inhibitors, genistein and tyrphostin 23, caused a rapid and specific fall in the cellular PtdIns(4,5)P2 level and prevented the stimulatory effect of pervanadate on PtdIns(4,5)P2 formation. Inactivation of Rho proteins by Clostridium difficile toxin B caused a similar fall in the HEK-293 cell PtdIns(4,5)P2 level, which was not altered by additional genistein treatment. Furthermore, toxin B treatment abolished the pervanadate-induced increase in PtdIns(4,5)P2 levels. As PtdIns(4,5)P2 is an essential stimulatory cofactor for phospholipase D (PLD) enzymes, we finally examined the effects of the agents regulating PtdIns(4,5)P2 levels on PLD activity in HEK-293 cells. Inhibition of tyrosine phosphatases by pervanadate caused an increase in PLD activity, which was susceptible to genistein and tyrphostin 23, and which was abolished by prior treatment with toxin B. In conclusion, the data presented indicate that the cellular level of the multifunctional phospholipid, PtdIns(4,5)P2, in HEK-293 cells is controlled by a tyrosine-kinase-dependent mechanism and that this process apparently involves Rho proteins, as found similarly for tyrosine-phosphorylation-induced PLD activation.
ESTHER : Rumenapp_1998_Biochem.J_334 ( Pt 3)_625
PubMedSearch : Rumenapp_1998_Biochem.J_334 ( Pt 3)_625
PubMedID: 9729471

Title : Sphingosine kinase-mediated Ca2+ signalling by G-protein-coupled receptors - Meyer_1998_EMBO.J_17_2830
Author(s) : Meyer zu Heringdorf D , Lass H , Alemany R , Laser KT , Neumann E , Zhang C , Schmidt M , Rauen U , Jakobs KH , van Koppen CJ
Ref : EMBO Journal , 17 :2830 , 1998
Abstract : Formation of inositol 1,4,5-trisphosphate (IP3) by phospholipase C (PLC) with subsequent release of Ca2+ from intracellular stores, is one of the major Ca2+ signalling pathways triggered by G-protein-coupled receptors (GPCRs). However, in a large number of cellular systems, Ca2+ mobilization by GPCRs apparently occurs independently of the PLC-IP3 pathway, mediated by an as yet unknown mechanism. The present study investigated whether sphingosine kinase activation, leading to production of sphingosine-1-phosphate (SPP), is involved in GPCR-mediated Ca2+ signalling as proposed for platelet-derived growth factor and FcepsilonRI antigen receptors. Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,N-dimethylsphingosine markedly inhibited [Ca2+]i increases elicited by m2 and m3 muscarinic acetylcholine receptors (mAChRs) expressed in HEK-293 cells without affecting mAChR-induced PLC stimulation. Activation of mAChRs rapidly and transiently stimulated production of SPP in HEK-293 cells. Finally, intracellular injection of SPP induced a rapid and transient Ca2+ mobilization in HEK-293 cells which was not antagonized by heparin. We conclude that mAChRs utilize the sphingosine kinase-SPP pathway in addition to PLC-IP3 to mediate Ca2+ mobilization. As Ca2+ signalling by various, but not all, GPCRs in different cell types was likewise attenuated by the sphingosine kinase inhibitors, we suggest a general role for sphingosine kinase, besides PLC, in mediation of GPCR-induced Ca2+ signalling.
ESTHER : Meyer_1998_EMBO.J_17_2830
PubMedSearch : Meyer_1998_EMBO.J_17_2830
PubMedID: 9582276

Title : Gi- and protein kinase C-mediated heterologous potentiation of phospholipase C signaling by G protein-coupled receptors - Schmidt_1998_Mol.Pharmacol_53_1139
Author(s) : Schmidt M , Lohmann B , Hammer K , Haupenthal S , Nehls MV , Jakobs KH
Ref : Molecular Pharmacology , 53 :1139 , 1998
Abstract : We recently reported that activation of the highly efficient phospholipase C (PLC) stimulatory m3 muscarinic acetylcholine receptor (mAChR) can induce a long-lasting Gi-mediated heterologous potentiation of PLC stimulation in human embryonic kidney (HEK) 293 cells, which was accompanied by an increased cellular level of the PLC substrate phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. Here, we examined whether such a potentiated PLC response is also induced by the rather poorly PLC stimulatory m2 mAChR and the endogenously expressed purinergic and lysophosphatidic acid receptors. Pretreatment of m2 mAChR-expressing HEK 293 cells for 2 min with carbachol, followed by agonist washout and measurement of PLC activity >/=40 min later, caused a long-lasting (up to approximately 90 min) heterologous potentiation of receptor- and G protein-mediated PLC stimulation. A similar heterologous potentiation of receptor-mediated PLC stimulation was induced by short term activation of lysophosphatidic acid and purinergic receptors. Either of the three receptor agonists increased the cellular level of PtdIns(4,5)P2 by approximately 50%. The mAChR-induced PLC potentiation was fully prevented by either pertussis toxin or the protein kinase C (PKC) inhibitors staurosporine and Go 6976, which did not affect acute PLC stimulation. On the other hand, the rise in PtdIns(4,5)P2 was prevented only by combined treatment of HEK 293 cells with pertussis toxin and PKC inhibitors. In conclusion, we demonstrated that activation of poorly PLC stimulatory receptors can also induce a long-lasting Gi-mediated heterologous potentiation of PLC signaling in HEK 293 cells and that this novel PLC regulatory process is under the control of PKC.
ESTHER : Schmidt_1998_Mol.Pharmacol_53_1139
PubMedSearch : Schmidt_1998_Mol.Pharmacol_53_1139
PubMedID: 9614219

Title : Specific inhibition of phorbol ester-stimulated phospholipase D by Clostridium sordellii lethal toxin and Clostridium difficile toxin B-1470 in HEK-293 cells. Restoration by Ral GTPases - Schmidt_1998_J.Biol.Chem_273_7413
Author(s) : Schmidt M , Voss M , Thiel M , Bauer B , Grannass A , Tapp E , Cool RH , de Gunzburg J , von Eichel-Streiber C , Jakobs KH
Ref : Journal of Biological Chemistry , 273 :7413 , 1998
Abstract : Activation of m3 muscarinic acetylcholine receptor (mAChR), stably expressed in human embryonic kidney (HEK)-293 cells, leads to phospholipase D (PLD) stimulation, a process apparently involving Rho GTPases, as shown by studies with Clostridium botulinum C3 exoenzyme and Clostridium difficile toxin B (TcdB). Direct activation of protein kinase C (PKC) by phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), also induces PLD stimulation, which is additive to the mAChR action and which is only poorly sensitive to inactivation of Rho proteins by TcdB. To study whether Ras-like GTPases are involved in PLD regulation, we studied the effects of the TcdB variant TcdB-1470 and Clostridium sordellii lethal toxin (TcsL), known to inactivate Rac and some members of the Ras protein family, on PLD activities. TcdB-1470 and TcsL did not affect basal PLD activity and PLD stimulation by mAChR or direct G protein activation. In contrast, PMA-induced PLD stimulation was inhibited by TcdB-1470 and TcsL in a time- and concentration-dependent manner, without alteration in immunologically detectable PKC isozyme levels. In membranes of HEK-293 cells pretreated with TcdB-1470 or TcsL, basal and stable GTP analog-stimulated PLD activities measured with exogenous phosphatidylcholine, in the presence or absence of phosphatidylinositol 4,5-bisphosphate, were not altered. In contrast, pretreatment with TcdB-1470 and TcsL, but not TcdB, strongly reduced PMA-stimulated PLD activity. The addition of recombinant Rac1, serving as glucosylation substrate for TcdB, TcsL, and TcdB-1470, did not restore PLD stimulation by PMA. Furthermore, PMA-stimulated PLD activity, suppressed by prior treatment with TcdB-1470 or TcsL, was not rescued by the addition of recombinant Ras (RasG12V) or Rap proteins, acting as glucosylation substrates for TcsL only (Ras) or TcdB-1470 and TcsL (Rap). In contrast, the addition of recombinant Ral proteins (RalA and RalB), glucosylation substrates for TscL and TcdB-1470, but not for TcdB, to membranes of TcdB-1470- or TcsL-treated cells fully restored PLD stimulation by PMA without altering the strict MgATP dependence of PMA-induced PLD stimulation. RalA-mediated restoration of PMA-stimulated PLD activity in membranes of TcsL-treated cells was not enhanced by coaddition of RasG12V. In conclusion, the data presented indicate that TcdB-1470 and TcsL selectively interfere with phorbol ester stimulation of PLD and suggest an essential role of Ral proteins in PKC signaling to PLD in HEK-293 cells.
ESTHER : Schmidt_1998_J.Biol.Chem_273_7413
PubMedSearch : Schmidt_1998_J.Biol.Chem_273_7413
PubMedID: 9516439

Title : Muscarinic receptor-stimulated cytosol-membrane translocation of RhoA - Keller_1997_FEBS.Lett_403_299
Author(s) : Keller J , Schmidt M , Hussein B , Rumenapp U , Jakobs KH
Ref : FEBS Letters , 403 :299 , 1997
Abstract : Receptor-mediated phospholipase D activation in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR) apparently involves the small G protein RhoA. Here, activation of RhoA was examined by measuring cytosol-membrane translocation, which is a sign of RhoA activation. RhoA translocation was induced by guanosine 5'-O-(3-thio)triphosphate in digitonin-permeabilized HEK cells, and in intact cells by the agonist-activated mAChR and by direct activation of heterotrimeric G proteins. RhoA translocation was also induced by the phosphotyrosine phosphatase inhibitor pervanadate, while the tyrosine kinase inhibitors tyrphostin 23 and genistein inhibited the mAChR-induced RhoA translocation. These data suggest that translocation and thus activation of RhoA by the G protein-coupled m3 mAChR in HEK cells apparently involves a tyrosine kinase-dependent reaction.
ESTHER : Keller_1997_FEBS.Lett_403_299
PubMedSearch : Keller_1997_FEBS.Lett_403_299
PubMedID: 9091321

Title : Regulation of phospholipase C and D activities by small molecular weight G proteins and muscarinic receptors - Schmidt_1997_Life.Sci_60(13-14)_1093
Author(s) : Schmidt M , Rumenapp U , Keller J , Lohmann B , Jakobs KH
Ref : Life Sciences , 60 :1093 , 1997
Abstract : The role of small molecular weight guanine nucleotide-binding proteins (G proteins) of the Rho family in muscarinic acetylcholine receptor (mAChR) signaling to phospholipase C (PLC) and phospholipase D (PLD) was studied in human embryonic kidney (HEK) cells, stably expressing the human m3 receptor subtype. Evidence for the involvement of Rho proteins in m3 mAChR signaling to both phospholipases is based on findings obtained with Clostridium (C.) difficile toxin B and C. botulinum C3 exoenzyme, both of which specifically, although by different mechanisms, inactivate Rho family G proteins. Toxin B potently inhibited both the mAChR-stimulated PLC and PLD activities in intact cells as well as the stimulation of both phospholipases by the stable GTP analog GTPgammaS in permeabilized cells, the latter effect being mimicked by C3 exoenzyme. In contrast, PLC and PLD activities, measured in the presence of exogenous phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], a substrate and cofactor for PLC and PLD, respectively, were not altered. These data suggested that the Rho-inactivating toxins inhibit stimulation of PLC and PLD by reducing the cellular level of PtdIns(4,5)P2, which was indeed found with both toxin B and C3 exoenzyme. In agreement with a crucial role of cellular PtdIns(4,5)P2 supply for PLC signaling, we observed that short-term agonist (carbachol) treatment of HEK cells caused a long-lasting increase in PtdIns(4,5)P2 level, accompanied by a potentiation of receptor- and G protein-stimulated inositol phosphate formation. Finally, studies with tyrosine kinase and tyrosine phosphatase inhibitors strongly suggest that PtdIns(4,5)P2 synthesis and mAChR-stimulated PLD activity in HEK cells apparently also involve a tyrosine phosphorylation-dependent mechanism(s). Thus, m3 mAChR signaling to PLC and PLD in HEK cells requires the concerted action of various intracellular components, most notably the complex regulation of PtdIns(4,5)P2 synthesis.
ESTHER : Schmidt_1997_Life.Sci_60(13-14)_1093
PubMedSearch : Schmidt_1997_Life.Sci_60(13-14)_1093
PubMedID: 9121352

Title : A role for Rho in receptor- and G protein-stimulated phospholipase C. Reduction in phosphatidylinositol 4,5-bisphosphate by Clostridium difficile toxin B - Schmidt_1996_Naunyn.Schmiedebergs.Arch.Pharmacol_354_87
Author(s) : Schmidt M , Bienek C , Rumenapp U , Zhang C , Lummen G , Jakobs KH , Just I , Aktories K , Moos M , von Eichel-Streiber C
Ref : Naunyn Schmiedebergs Arch Pharmacol , 354 :87 , 1996
Abstract : Receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins) activate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-hydrolyzing phospholipase C (PLC) enzymes by activated alpha of free beta gamma subunits of the relevant G proteins. To study whether low molecular weight G proteins of the Rho family are involved in receptor signaling to PLC, we examined the effect of Clostridium difficile toxin B, which glucosylates and thereby inactivates Rho proteins, on the regulation of PLC activity in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR) subtype. Toxin B treatment of HEK cells did not affect basal PLC activity, but potently and efficiently inhibited mAChR-stimulated inositol phosphate formation. PLC activation by the endogenously expressed thrombin receptor and by the direct G protein activators, A1F-4 and guanosine 5'-[gamma-thio]triphosphate (GTP gamma S), studied in intact and permeabilized cells, respectively, were also inhibited by toxin B treatment. C3 exoenzyme, which ADP-ribosylates Rho proteins, mimicked the inhibitory effect of toxin B on GTP gamma S-stimulated PLC activity. Finally both toxin B and C3 exoenzyme significantly reduced, by 40 to 50%, the total level of PtdIns(4,5)P2 in HEK cells, without affecting the levels of phosphatidylinositol and phosphatidylinositol 4-phosphate. Accordingly, When PLC activity was measured with exogenous PtdIns(4,5)P2 as enzyme substrate, Ca(2+)- as well as GTP gamma S- or A1F-4-stimulated PLC activities were not altered by prior toxin B treatment. In conclusion, evidence is provided that toxin B and C3 exoenzyme, apparently by inactivating Rho proteins, inhibit G protein-coupled receptor signalling to PLC, most likely by reducing the cellular substrate supply.
ESTHER : Schmidt_1996_Naunyn.Schmiedebergs.Arch.Pharmacol_354_87
PubMedSearch : Schmidt_1996_Naunyn.Schmiedebergs.Arch.Pharmacol_354_87
PubMedID: 8857584

Title : Participation of small GTP-binding proteins in m3 muscarinic acetylcholine receptor signalling to phospholipase D and C -
Author(s) : Rumenapp U , Schmidt M , Geiszt M , Jakobs KH
Ref : Prog Brain Res , 109 :209 , 1996
PubMedID: 9009709

Title : m3 Muscarinic receptor-induced and Gi-mediated heterologous potentiation of phospholipase C stimulation: role of phosphoinositide synthesis - Schmidt_1996_Mol.Pharmacol_50_1038
Author(s) : Schmidt M , Nehls C , Rumenapp U , Jakobs KH
Ref : Molecular Pharmacology , 50 :1038 , 1996
Abstract : Agonist activation of thrombin and purinergic receptors endogenously expressed in human embryonic kidney (HEK) cells and of the stably expressed m3 muscarinic acetylcholine receptor (mAChR) induces phospholipase C (PLC) stimulation, with the most pronounced PLC stimulation observed on mAChR activation. These receptor responses were pertussis toxin (PTX) insensitive and nonadditive, suggesting that the receptors share common signaling pathways. Short term (2 min) pretreatment of HEK cells with carbachol (1 mM), but not ATP, followed by agonist washout, caused a long-lasting (> or = 90 min) sensitization of PLC responses. At 30 min after carbachol treatment and washout, mAChR-stimulated PLC activity, measured as formation of either total inositol phosphates or of inositol-1,4,5-trisphosphate, was enhanced by 1.5-2-fold. PLC stimulation by thrombin and purinergic receptors was increased by approximately 3-fold. Furthermore, carbachol pretreatment also enhanced, by approximately 2.5-fold, stimulation of PLC activity on direct activation of G proteins by AIF4- and guanosine-5'-O-(3-thio)-triphosphate in intact and permeabilized cells, respectively. In contrast, PLC activities, measured with exogenous phosphatidylinositol-4,5-bisphosphate [Ptdns(4,5)P2] in HEK cell lysates, were not altered, suggesting that carbachol pretreatment may enhance the cellular level of Ptdlns(4,5)P2. Indeed, the level of Ptdlns(4,5)P2 was found to be increased by approximately 50% in HEK cells 30 min after short term carbachol treatment, whereas the level of phosphatidylinositol was not altered and that of phosphatidylinositol-4-phosphate decreased (by 40-50%). Pretreatment of HEK cells with PTX prevented the m3 mAChR-induced PLC potentiation and reduced the elevation in Ptdlns(4,5)P2 level by approximately 50%. In conclusion, short term agonist activation of m3 mAChRs stably expressed in HEK cells can lead to a longlasting heterologous potentiation of PLC signaling, which processes apparently involve PTX-sensitive G proteins and an enhanced PLC substrate supply.
ESTHER : Schmidt_1996_Mol.Pharmacol_50_1038
PubMedSearch : Schmidt_1996_Mol.Pharmacol_50_1038
PubMedID: 8863852

Title : Inhibition of receptor signaling to phospholipase D by Clostridium difficile toxin B. Role of Rho proteins - Schmidt_1996_J.Biol.Chem_271_2422
Author(s) : Schmidt M , Rumenapp U , Bienek C , Keller J , von Eichel-Streiber C , Jakobs KH
Ref : Journal of Biological Chemistry , 271 :2422 , 1996
Abstract : Rho proteins have been reported to activate phospholipase D (PLD) in in vitro preparations. To examine the role of Rho proteins in receptor signaling to PLD, we studied the effect of Clostridium difficile toxin B, which glucosylates Rho proteins, on the regulation of PLD activity in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR). Toxin B treatment of HEK cells potently and efficiently blocked mAChR-stimulated PLD. In contrast, basal and phorbol ester-stimulated PLD activities were not or only slightly reduced. Cytochalasin B and Clostridium botulinum C2 toxin, mimicking the effect of toxin B on the actin cytoskeleton but without involving Rho proteins, had no effect on mAChR-stimulated PLD. Toxin B did not alter cell surface mAChR number and mAChR-stimulated binding of (guanosine 5'-O-(thio)triphosphate (GTP gamma S) to G proteins. In addition to mAChR-stimulated PLD, toxin B treatment also inhibited PLD activation by the direct G protein activators, AlF4- and GTP gamma S, studied in intact and permeabilized cells, respectively. Finally, C. botulinum C3 exoenzyme, which ADP-ribosylates Rho proteins, mimicked the inhibitory effect of toxin B on GTP gamma S-stimulated PLD activity. In conclusion, the data presented indicate that toxin B potently and selectively interferes with receptor coupling mechanisms to PLD, and furthermore suggest an essential role for Rho proteins in receptor signaling to PLD.
ESTHER : Schmidt_1996_J.Biol.Chem_271_2422
PubMedSearch : Schmidt_1996_J.Biol.Chem_271_2422
PubMedID: 8576201

Title : Restoration of Clostridium difficile toxin-B-inhibited phospholipase D by phosphatidylinositol 4,5-bisphosphate - Schmidt_1996_Eur.J.Biochem_240_707
Author(s) : Schmidt M , Rumenapp U , Nehls C , Ott S , Keller J , von Eichel-Streiber C , Jakobs KH
Ref : European Journal of Biochemistry , 240 :707 , 1996
Abstract : Receptor signalling to phospholipase D (PLD) in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor apparently involves Rho proteins. Since phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has been recognized as an essential cofactor for PLD activity and since activated Rho proteins have been reported to stimulate the synthesis of PtdIns(4,5)P2, we studied whether in HEK cells PLD activity is regulated by PtdIns(4,5)P2 and, in particular, whether PtdIns(4,5)P2 can restore PLD activity inhibited by Clostridium difficile toxin B, which inactivates Rho proteins. Addition of MgATP to permeabilized HEK cells increased basal PLD activity and potentiated PLD stimulation by the stable GTP analogue, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), concomitant with a large increase in PtdIns(4,5)P2. On the other hand, neomycin, which binds to PtdIns(4,5)P2, inhibited basal and GTP[S]-stimulated PLD activities. Addition of PtdIns(4,5)P2 increased PLD activity in HEK cell membranes by 2-3-fold, whereas various other phospholipids were ineffective. Prior treatment of HEK cells with toxin B reduced the level of PtdIns(4,5)P2, measured either in intact cells or in membrane preparations, by about 40%. In membranes of toxin-B-treated cells, basal and GTP[S]-stimulated PLD activities were reduced, when measured with exogenous phosphatidylcholine as enzyme substrate. Inclusion of PtdIns(4,5)P2 with phosphatidylcholine in the substrate vesicles or addition of PtdIns(4,5)P2 fully restored basal and GTP[S]-stimulated PLD activities in membranes of toxin-B-treated cells. In conclusion, the data indicate that PtdIns(4,5)P2 is an essential cofactor for PLD activity in HEK cells and that inhibition of PLD activity by the Rho-inactivating toxin B is apparently caused by depletion of the PLD cofactor, PtdIns(4,5)P2.
ESTHER : Schmidt_1996_Eur.J.Biochem_240_707
PubMedSearch : Schmidt_1996_Eur.J.Biochem_240_707
PubMedID: 8856074

Title : Evidence for ADP-ribosylation-factor-mediated activation of phospholipase D by m3 muscarinic acetylcholine receptor - Rumenapp_1995_Eur.J.Biochem_234_240
Author(s) : Rumenapp U , Geiszt M , Wahn F , Schmidt M , Jakobs KH
Ref : European Journal of Biochemistry , 234 :240 , 1995
Abstract : Activation of phospholipase D (PLD) is a cellular response to a wide variety of extracellular ligands. However, the exact mechanisms that link cell surface receptors to PLD remain unclear. In this study, we report the involvement of the small-molecular-mass guanine-nucleotide-binding protein, ADP-ribosylation factor (ARF), in the activation of PLD by the muscarinic acetylcholine receptor (mAChR) in human embryonic kidney cells stably expressing the human m3 subtype. PLD stimulation in permeabilized cells by guanosine 5'-O-[gamma-thio]triphosphate (GTP[S]) was dependent on a cytosolic factor and reconstituted by purified recombinant ARF 1. Brefeldin A, a known inhibitor of the ARF guanine-nucleotide-exchange-factor activity in Golgi membranes, inhibited mAChR-stimulated PLD, whereas basal PLD activity and stimulation by GTP[S] were not affected. Upon cell permeabilization without the addition of stimulus, ARF proteins were released. However, the addition of GTP[S] during permeabilization and mAChR activation before permeabilization caused an almost complete and partial (about 60%) inhibition, respectively, of ARF release, indicating that ARF proteins are activated and thereby translocated to membranes. The results indicate that ARF proteins and their nucleotide-exchange factor are apparently involved in the signalling pathway leading from mAChR activation to PLD stimulation in human embryonic kidney cells.
ESTHER : Rumenapp_1995_Eur.J.Biochem_234_240
PubMedSearch : Rumenapp_1995_Eur.J.Biochem_234_240
PubMedID: 8529647

Title : The role of membrane proximal threonine residues conserved among guanine-nucleotide-binding-protein-coupled receptors in internalization of the m4 muscarinic acetylcholine receptor - Van Koppen_1995_Eur.J.Biochem_234_536
Author(s) : van Koppen CJ , Lenz W , Nunes JP , Zhang C , Schmidt M , Jakobs KH
Ref : European Journal of Biochemistry , 234 :536 , 1995
Abstract : Many guanine-nucleotide-binding-protein-coupled receptors contain consensus sequences for phosphorylation by cAMP-dependent protein kinase (PKA), often located in the membrane proximal regions critically important for receptor signalling. In the present study, we have evaluated by site-directed mutagenesis the role of the putative PKA phosphorylation sites in the m4 muscarinic acetylcholine receptor (mAChR), i.e. Thr145 in the second cytoplasmic loop and Thr399 in the third cytoplasmic loop, and the influence of PKA on m4 mAChR function and internalization. Antagonist binding was unaltered by any of the mutations studied, while the agonist-binding affinity was either not affected (Thr145 alanine), increased (Thr399 alanine) or decreased (Thr399 serine or aspartic acid). m4 mAChR-mediated inhibition of adenylyl cyclase was unaltered by the mutations, except for an approximately tenfold reduced agonist potency of the Thr399 aspartic acid mutated receptor. Agonist-induced receptor internalization was unaltered with Thr399 serine or aspartic acid mutations of the receptors, but was strongly decreased in its rate and extent upon replacement of Thr399, Thr145 or both of these residues with alanine. These mutational effects could not be reproduced by treatment of wild-type receptor-expressing cells with the PKA inhibitor H-8. Furthermore, maximal stimulation of cellular PKA neither affected receptor internalization nor signalling measured as receptor-mediated Ca2+ mobilization. We conclude that the membrane proximal threonine residues of the m4 mAChR are not required for receptor signalling, but replacement by alanine residues can significantly affect receptor internalization, independently of PKA phosphorylation. Sequence comparisons suggest that threonine residues at corresponding positions may be relevant to internalization of other guanine-nucleotide-binding-protein-coupled receptors.
ESTHER : Van Koppen_1995_Eur.J.Biochem_234_536
PubMedSearch : Van Koppen_1995_Eur.J.Biochem_234_536
PubMedID: 8536700

Title : Differential calcium signalling by m2 and m3 muscarinic acetylcholine receptors in a single cell type - Schmidt_1995_Naunyn.Schmiedebergs.Arch.Pharmacol_352_469
Author(s) : Schmidt M , Bienek C , van Koppen CJ , Michel MC , Jakobs KH
Ref : Naunyn Schmiedebergs Arch Pharmacol , 352 :469 , 1995
Abstract : We have compared muscarinic acetylcholine receptor (mAChR) coupling to phospholipase C (PLC) and increases in cytoplasmic Ca2+ concentration [Ca2+]i in human embryonic kidney (HEK) cells, stably expressing either the human m3 or m2 receptor subtype. In m3 mAChR-expressing cells, carbachol stimulated inositol phosphate (InsP) formation and increased [Ca2+]i with EC50 values of about 2 microM and 30 nM, respectively. Maximal inositol 1,4,5-trisphosphate (InsP3) production (about fourfold) was rapid (15 s) and stable for 2 min. Maximal increases in [Ca2+]i were 300-350 nM and mainly, almost 90%, due to influx of extracellular Ca2+. The efficacy of pilocarpine for stimulating InsP and Ca2+ responses was not significantly different from that of carbachol. All m3 mAChR-mediated responses were pertussis toxin (PTX)-insensitive. In m2 mAChR-expressing cells, carbachol stimulated InsP formation and increased [Ca2+]i with EC50 values of about 20 microM and 7 microM, respectively. Maximal InsP formation was only 10-15% of that observed in m3 mAChR-expressing cells, whereas maximal elevations of [Ca2+]i were similar in both cell types. Formation of InsP3 was rapid (15 s to 2 min) and about twofold above basal. In contrast to m3 mAChR activation, [Ca2+]i increases induced by m2 mAChR activation were exclusively due to Ca2+ mobilization from intracellular stores. The efficacy of pilocarpine for stimulating InsP and Ca2+ responses was 50% and 20% of the efficacy of carbachol, respectively. PTX treatment did not affect m2 mAChR-induced PLC stimulation, but reduced the m2 mAChR-mediated increases in [Ca2+]i to 50%. In conclusion, m3 and m2 mAChRs stably expressed in HEK cells can induce similar cellular responses; however, they do so by activating apparently distinct signalling pathways. While coupling of m2 mAChR to PLC occurs in a PTX-insensitive manner, coupling to mobilization of Ca2+ from intracellular stores is partly PTX-sensitive and this may occur at least partly independent of PLC activation.
ESTHER : Schmidt_1995_Naunyn.Schmiedebergs.Arch.Pharmacol_352_469
PubMedSearch : Schmidt_1995_Naunyn.Schmiedebergs.Arch.Pharmacol_352_469
PubMedID: 8751074

Title : Rapid and persistent desensitization of m3 muscarinic acetylcholine receptor-stimulated phospholipase D. Concomitant sensitization of phospholipase C - Schmidt_1995_J.Biol.Chem_270_19949
Author(s) : Schmidt M , Fasselt B , Rumenapp U , Bienek C , Wieland T , van Koppen CJ , Jakobs KH
Ref : Journal of Biological Chemistry , 270 :19949 , 1995
Abstract : Activation of muscarinic acetylcholine receptors (mAChR) in human embryonic kidney (HEK) cells stably expressing the human m3 subtype leads to stimulation of both phospholipase C (PLC) and D (PLD). mAChR-stimulated PLD was turned off after 2 min of receptor activation with either the full (carbachol) or partial agonist (pilocarpine) and remained completely suppressed for at least 4 h. Partial recovery was observed 24 h after agonist removal. This rapid arrest of PLD response was not due to a loss of cell surface receptors and was also not caused by negative feedback due to concomitant activation of protein kinase C, tyrosine phosphorylation, increase in cytosolic calcium, or activation of Gi proteins. Furthermore, PLD stimulation by directly activated protein kinase C and GTP-binding proteins was unaltered in carbachol-pretreated cells. Finally, neither prevention of PLD stimulation during carbachol pretreatment by genistein nor inhibition of protein synthesis by cycloheximide, added before or after carbachol challenge, resulted in recovery of mAChR-stimulated PLD. The short term carbachol pretreatment nearly completely abolished agonist-induced binding of guanosine 5'-O-(3-thiotriphosphate) to membranes or permeabilized adherent cells. Full recovery of this response was achieved after 4 h. Similar to transfected m3 mAChR, PLD stimulation by endogenously expressed purinergic receptors was also fully blunted after 2 min of agonist (ATP) treatment. Preexposure of HEK cells to either receptor agonist partially, but not completely, reduced PLD stimulation by the other agonist. In contrast to desensitization of PLD stimulation, 2 min of carbachol treatment led to a sensitization, by up to 2-fold, of mAChR-stimulated inositol phosphate formation. This supersensitivity was also observed with pilocarpine, which acted as a full agonist on PLC. On the basis of these results, we conclude that the m3 mAChR stimulates PLD and PLC in HEK cells with distinct efficiencies and with very distinct durations of each response. The rapid and long lasting desensitization of the PLD response is apparently not due to a loss of cell surface receptors or PLD activation by GTP-binding proteins, but it may involve, at least initially, an uncoupling of receptors from GTP-binding proteins and most likely a loss of an as yet undefined essential transducing component.
ESTHER : Schmidt_1995_J.Biol.Chem_270_19949
PubMedSearch : Schmidt_1995_J.Biol.Chem_270_19949
PubMedID: 7650010

Title : Analysis of receptor-G protein interactions in permeabilized cells - Wieland_1995_Naunyn.Schmiedebergs.Arch.Pharmacol_351_329
Author(s) : Wieland T , Liedel K , Kaldenberg-Stasch S , Meyer zu Heringdorf D , Schmidt M , Jakobs KH
Ref : Naunyn Schmiedebergs Arch Pharmacol , 351 :329 , 1995
Abstract : Receptor-induced binding of the stable GTP analogue, guanosine 5'-[gamma-thio]triphosphate (GTP [gamma S]), to guanine nucleotide-binding regulatory proteins (G proteins) was measured in various permeabilized cells. In myeloid differentiated human leukemia (HL-60) cells, permeabilized with either digitonin, streptolysin O or Staphylococcus aureus alpha-toxin, binding of GTP[gamma S] induced by three distinct chemoattractant receptors was observed. The extent of receptor-stimulated GTP[gamma S] binding (maximally about 2-fold) was independent of the type of permeabilizing agent used. In human erythroleukemia cells permeabilized with digitonin, agonist activation of thrombin and neuropeptide Y receptors increased GTP[gamma S] binding by 1.8- and 1.5-fold, respectively. Finally, in adherently grown human embryonic kidney cells permeabilized with digitonin, activation of the stably expressed human muscarinic m3 receptor increased GTP[gamma S] binding by about 1.6-fold. In digitonin-permeabilized HL-60 cells, a quantitative analysis of formyl peptide receptors and interacting G proteins was performed. About 50,000 formyl peptide receptors per cell were detected. Agonist binding to these receptors was fully sensitive to regulation by guanine nucleotides and pertussis toxin. The number of high-affinity GTP[gamma S] binding sites, most likely representing heterotrimeric G proteins, was calculated to be about 670,000 per cell. Stimulation of formyl peptide receptors led to the activation of about 130,000 of high-affinity GTP[gamma S] binding sites, indicating a ratio of about three activated G proteins per one agonist-activated receptor.(ABSTRACT TRUNCATED AT 250 WORDS)
ESTHER : Wieland_1995_Naunyn.Schmiedebergs.Arch.Pharmacol_351_329
PubMedSearch : Wieland_1995_Naunyn.Schmiedebergs.Arch.Pharmacol_351_329
PubMedID: 7630424

Title : Mechanisms of phospholipase D stimulation by m3 muscarinic acetylcholine receptors. Evidence for involvement of tyrosine phosphorylation - Schmidt_1994_Eur.J.Biochem_225_667
Author(s) : Schmidt M , Huwe SM , Fasselt B , Homann D , Rumenapp U , Sandmann J , Jakobs KH
Ref : European Journal of Biochemistry , 225 :667 , 1994
Abstract : In human embryonic kidney cells stably expressing the human m3 muscarinic acetylcholine receptor (mAChR) subtype, agonist (carbachol) activation stimulated phospholipase C, increased cytoplasmic calcium concentration, induced tyrosine phosphorylation of various cellular proteins and activated phospholipase D. Bypassing membrane receptors, phospholipase D was activated in these cells by direct activation of protein kinase C by phorbol esters, by direct activation of GTP-binding proteins by A1F4- and a stable GTP analogue (in permeabilized cells), by increasing cytoplasmic calcium concentration with the calcium ionophore A23187 and also apparently by tyrosine phosphorylation. In order to identify possible mechanisms by which the m3 mAChR couples to phospholipase D, various inhibitors of protein kinase C, tyrosine kinases and calcium-dependent events were studied. Prevention of an agonist-induced increase in cytoplasmic calcium concentration did not alter the mAChR-induced phospholipase D stimulation. The protein kinase C inhibitors, calphostin C and staurosporine, efficiently prevented phospholipase D activation by phorbol 12-myristate 13-acetate but only partially inhibited the activation induced by the mAChR agonist. Additionally, down-regulation of protein kinase C by prolonged exposure to phorbol 12-myristate 13-acetate abrogated phospholipase D activation by this effector but had only minor or no effects on the response to the mAChR agonist and direct activators of GTP-binding proteins. In contrast, the tyrosine kinase inhibitor genistein abolished the carbachol-induced and A1F4(-)-induced phospholipase D activation but had no effect on enzyme activation by phorbol 12-myristate 13-acetate. The data indicate that phospholipase D in m3 mAChR-expressing human embryonic kidney cells can be activated by various different mechanisms, i.e. receptor agonists, GTP-binding proteins, protein kinase C-dependent and calcium-dependent events and tyrosine phosphorylation. The coupling of m3 mAChR to phospholipase D appears to be largely independent of concomitant phospholipase C activation with subsequent increase in cytoplasmic calcium concentration and protein kinase C activity. The data instead suggest the involvement of an essential protein tyrosine phosphorylation mechanism in phopsholipase D activation by the m3 mAChR and heterotrimeric GTP-binding proteins.
ESTHER : Schmidt_1994_Eur.J.Biochem_225_667
PubMedSearch : Schmidt_1994_Eur.J.Biochem_225_667
PubMedID: 7957182

Title : Response of myogenic determination factors to cessation and resumption of electrical activity in skeletal muscle: a possible role for myogenin in denervation supersensitivity - Neville_1992_Cell.Mol.Neurobiol_12_511
Author(s) : Neville CM , Schmidt M , Schmidt J
Ref : Cellular Molecular Neurobiology , 12 :511 , 1992
Abstract : 1. We have prepared probes specific for the chicken myogenic determination genes MyoD, myogenin, myf5, and herculin and have investigated the expression of these genes in response to denervation and acute electrical stimulation in neonate chick muscle, using ribonuclease protection. 2. Upon denervation, herculin mRNA remains essentially unchanged, myf5 transcript levels approximately double, and MyoD message is up-regulated by two- to fivefold. In contrast, the message coding for myogenin, barely detectable in innervated muscle, rises dramatically (approximately 200-fold) on the second day after nerve section; in this respect it resembles acetylcholine receptor (AChR) alpha-, gamma- and delta-subunit mRNAs. Cohybridization experiments reveal that the increase in myogenin mRNA slightly precedes the rise in AChR alpha-subunit message. 3. Electrical stimulation of denervated muscle leads to an immediate decline in myogenin and AChR alpha-subunit mRNAs, with half-lives of less than an hour and approximately 4 hr, respectively; message stability measurements suggest that this is effected through a rapid shutdown of transcription. Messages coding for MyoD, myf5, and herculin decay much more slowly, as a result of slower turnover. 4. Previous experiments have indicated the involvement of a de novo induced (Tsay, H.-J., Neville, C. M., and Schmidt, J., FEBS Lett. 274:69-72, 1990) autocatalytic (Neville, C. M., Schmidt, M., and Schmidt, J., NeuroReport 2:655-657, 1991) transcription factor in the denervation-triggered up-regulation of AChR alpha-subunit expression; the denervation and electrical stimulation experiments reported here are compatible with the notion that myogenin is that factor.
ESTHER : Neville_1992_Cell.Mol.Neurobiol_12_511
PubMedSearch : Neville_1992_Cell.Mol.Neurobiol_12_511
PubMedID: 1337017

Title : Kinetics of expression of ACh receptor alpha-subunit mRNA in denervated and stimulated muscle - Neville_1991_Neuroreport_2_655
Author(s) : Neville C , Schmidt M , Schmidt J
Ref : Neuroreport , 2 :655 , 1991
Abstract : Levels of the acetylcholine receptor (AChR) alpha-subunit mRNA were quantified in chick leg muscle, both after section of the sciatic nerve and following electrical stimulation of the denervated leg musculature. Whereas a lag period of approximately 17 h intervenes between the nerve section and the increase in message level, electrical stimulation leads to an immediate decline, which proceeds with a half-life of 3-4 h, similar to the decay induced by treatment with actinomycin D. The asymmetry in the kinetics of activation and repression can be accommodated by several regulatory schemes of which the simplest contains an autocatalytic loop as recently proposed by Changeux (The New Biologist 3: 413-429, 1991).
ESTHER : Neville_1991_Neuroreport_2_655
PubMedSearch : Neville_1991_Neuroreport_2_655
PubMedID: 1810458

Title : Proceedings: Inhibition of acetylcholinesterase in the bronchial system of rats caused by inhalation of dichlorvos -
Author(s) : Schmidt G , Schmidt M , Nenner M
Ref : Naunyn Schmiedebergs Arch Pharmacol , 287 Suppl :R97 , 1975
PubMedID: 1143525